Kit for enhancing the association rates of polynucleotides

ABSTRACT

Kit for increasing the association rate between a polynucleotide probe and a target nucleic acid, where the kit includes a polynucleotide probe, a synthetic polycationic polymer for increasing the association rate of the probe and a target nucleic acid, and a dissociating reagent for dissociating the polymer from the probe and the target nucleic acid.

[0001] This application is a divisional of application Ser. No.10/020,596, filed Dec. 7, 2001, now pending, the contents of which arehereby incorporated by reference herein, which claims the benefit ofU.S. Provisional Application No. 60/255,535, filed Dec. 14, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to a method and kit for use informing duplexes from single-stranded, complementary regions ofpolynucleotides which include one or more synthetic, water solublepolycationic polymers which enhance the association rates of thepolynucleotides under assay conditions.

INCORPORATION BY REFERENCE

[0003] All references referred to herein are hereby incorporated byreference in their entirety. The incorporation of these references,standing alone, should not be construed as an assertion or admission bythe inventors that any portion of the contents of all of thesereferences, or any particular reference, is considered to be essentialmaterial for satisfying any national or regional statutory disclosurerequirement for patent applications. Notwithstanding, the inventorsreserve the right to rely upon any of such references, whereappropriate, for providing material deemed essential to the claimedinvention by an examining authority or court. No reference referred toherein is admitted to be prior art to the claimed invention.

BACKGROUND OF THE INVENTION

[0004] Nucleic acid association concerns techniques by whichcomplementary base regions of two separate strands of nucleic acid, ordistinct base regions of the same nucleic acid strand, anneal to eachother through Watson-Crick base pairing, thus forming at least partiallydouble-stranded nucleic acids. Examples of such pairing include twocomplementary DNA sequences, a single-stranded DNA sequence and acomplementary RNA sequence, and two complementary RNA sequences, as wellas modified forms of these nucleic acids, such as peptide nucleic acidsor locked nucleic acids. See Nielsen et al, “Peptide Nucleic Acids,”U.S. Pat. No. 5,773,571; see also Imanishi et al., “Bicyclonucleosideand Oligonucleotide Analogues,” U.S. Pat. No. 6,268,490. For basepairing to take place, it is necessary to incubate the single-strandednucleic acids under conditions which facilitate the formation of stableduplexes between nucleic acids or within a nucleic acid havingcomplementary base regions. The formation of duplexes under stringentassay conditions can provide useful information about at least onemember of each duplex, including the source from which that particularmember was derived (e.g., a specific virus, microorganism, plant oranimal), whether directly or by nucleic acid amplification, the presenceof a disease-associated gene, the level of gene expression, theidentification of genetic variability, including the detection ofmutations and polymorphisms, and nucleic acid sequence information.

[0005] The rate at which nucleic acids having complementary base regionsassociate to form duplexes follows second-order kinetics. Thus, as theconcentration of single-stranded nucleic acids is increased, thereaction rate (i.e., the rate at which duplexes are formed) is alsoincreased. Conversely, as the concentration of single-stranded nucleicacids is decreased, the reaction rate is likewise decreased and, as aresult, more time is required for the formation of double-strandednucleic acids.

[0006] It is also well known that the temperature a reaction mixtureaffects the rate at which complementary nucleic acids associate. Forexample, as the temperature of a reaction mixture falls below themelting temperature or T_(m) (the temperature at which 50% of theduplexed molecules are rendered single-stranded), a maximum rate ofreaction will be reached at temperatures approximately 15° to 30° C.below the T_(m). Decreasing the temperature still further will lower thereaction rate below this maximum rate.

[0007] The reaction rate between nucleic acids having complementary baseregions is also very dependent upon the ionic strength of a reactionmixture. Deoxynucleic acids, for instance, show a marked increase inreaction rates up to about 1.2 M NaCl, at which point the rate ofassociation becomes essentially constant. See Wetmur et al. J. Mol.Biol. (1968) 31:349-379. And hybridizations between DNA and RNAmolecules show a 5-6 fold increase in the rate of association when theionic strength is increased from 0.2 to 1.5 M NaCl. The effect ofchanges in salt concentration on the rate of DNA:DNA reactions isgreater than it ifor RNA:DNA reactions.

[0008] A major limitation on the utility of many known nucleic acidassociation techniques is the basic rate of the reaction. Reaction timescan be on the order of several hours to tens of hours, and even days incertain instances. Increasing the reaction rate by increasing the amountof single-stranded nucleic acid molecules in a reaction, in order totake advantage of second-order kinetics, is an undesirable solution forseveral reasons. First, in many applications the targeted nucleic acidin a reaction mixture has been extracted from a physiological sample,thereby imposing inherent limits on the amount of the targeted nucleicacid available in the reaction mixture, at least in the absence of anamplification procedure such as the polymerase chain reaction. See,e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S. Pat.No. 4,683,202. Second, there are considerable expenses associated withthe use of nucleic acid reactants, and even greater expenses associatedwith a nucleic acid amplification procedure (e.g., enzymes andamplification primers), thus affecting the practicality of using more ofthese reactants or adding amplification reactants. Third, increasing thequantity of labeled, single-stranded nucleic acid probe molecules in areaction mixture will cause a decrease in the sensitivity of a reaction,since the additional single-stranded nucleic acid probe molecules willelevate the background noise.

[0009] Accordingly, it is a principal object of the present invention toprovide a method for forming a duplex from single-stranded regions ofseparate polynucleotides which enhances the association rate of thepolynucleotides, whether reassociation or hybridization, and whichenhancement can be demonstrated using reference conditions, includingthose conditions set forth herein. It is a further object of the presentinvention to provide a method for enhancing association rates that wouldbe applicable to DNA:DNA, RNA:DNA and RNA:RNA reaction systems.Additionally, it is an object of the present invention to provide amethod for promoting polynucleotide association rates that is applicableto reaction mixtures having a range of temperature and ionic strengthconditions.

SUMMARY OF THE INVENTION

[0010] In satisfaction of these objectives, the present inventionfeatures a method for forming a duplex from a polynucleotide probe and atarget nucleic acid, where the method comprises the steps of: (i)providing the probe to a test sample under conditions permitting theprobe to preferentially hybridize to the target nucleic acid, if any,present in the sample; and (ii) providing a synthetic polycationicpolymer to the test sample in an amount sufficient to increase theassociation rate of the probe and the target nucleic acid in the sampleunder the conditions permitting the probe to preferentially hybridize tothe target nucleic acid. To facilitate detecting the formation ofprobe:target nucleic acid hybrids, this method may further compriseproviding to the test sample a reagent to dissociate the polymer fromthe probe in some detection systems. Additionally, this method may beused in an assay to determine the presence or absence of a targetnucleic acid sequence derived from a target virus or organism or atarget group of viruses or organisms as an indication of the presence orabsence of the target virus or organism or members of the target groupof viruses or organisms in the test sample. Alternative uses of thismethod include detecting the presence of a disease-associated gene,determining the state of a disease, measuring levels of gene expression,and detecting mutations or polymorphisms in a test sample.

[0011] In addition to anionic groups, polynucleotide probes featured inthe present invention may further include cationic and/or nonionicgroups, provided the probes have a net positive charge. Thepolynucleotide may consist of deoxyribonucleic acid (DNA), ribonucleicacid (RNA), a combination of DNA and RNA, or it may include a nucleicacid analog (e.g., a peptide nucleic acid) or contain one or moremodified nucleosides (e.g., a ribonucleoside having a 2′-O-methylsubstitution to the ribofuranosyl moiety). Non-nucleotide groups, suchas polysaccharides or polyethyelene glycol, may also be included in theprobes, provided they do not prevent or substantially interfere withhybridization of the probe to the target nucleic acid. Probes of thepresent invention are up to 100 bases or more in length (preferably from12 to 50 bases, and more preferably from 18 to 35 bases in length) andcontain a base region which is complementary to a target sequencecontained in the target nucleic acid (the base region is preferablyperfectly complementary to the target sequence).

[0012] The probes preferably include a detectable label or group ofinteracting labels. The label may be any suitable labeling substance,including but not limited to a radioisotope, an enzyme, an enzymecofactor, an enzyme substrate, a dye, a hapten, a chemiluminescentmolecule, a fluorescent molecule, a phosphorescent molecule, anelectrochemiluminescent molecule, a chromophore, a base sequence regionthat is unable to stably bind to the target nucleic acid under thestated conditions, and mixtures of these. In one particularly preferredembodiment, the label is an acridinium ester (AE), preferably4-(2-succinimidyloxycarbonylethyl)-phenyl-10-methylacridinium-9-carboxylate fluorosulfonate(hereinafter referred to as “standard AE”). Groups of interacting labelsinclude, but are not limited to, enzyme/substrate, enzyme/cofactor,luminescent/quencher, luminescent/adduct, dye dimers and Forresterenergy transfer pairs. When provided with a group of interacting labels,the probes preferably have regions of self-complementarity such that thegroup of interacting labels produce a first signal when an associatedprobe is self-hybridized and a second signal when the probe ishybridized to the target nucleic acid, where the first and secondsignals are differentially detectable. In some embodiments, theseregions of self-complementarity may overlap or include the portion orportions of the probe which hybridize to the target nucleic acid.

[0013] To isolate the target nucleic acid in a test sample prior todetection, the present invention further contemplates probes having abase sequence region distinct from the target binding region of theprobe which constitutes an immobilized probe binding region of a captureprobe. The immobilized probe binding region may be comprised of, forexample, a 3′ poly dA (adenine) region which hybridizes under stringentconditions to a 5′ poly dT (thymine) region of a polynucleotide bounddirectly or indirectly to a solid support provided to the test sample.Any known solid support may be used, such as matrices and particles freein solution. The solid support may be, for example, nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and, preferably, particles having a magnetic charge tofacilitate recovering sample and/or removing unbound nucleic acids orother sample components. Particularly preferred supports are magneticspheres that are monodisperse (i.e., uniform in size±5%), allowing forconsistent results, which is particularly advantageous for use in anautomated procedure.

[0014] The target nucleic acid may be RNA or DNA, a nucleic acid analogor a chimeric containing different types of nucleic acid and/or nucleicacid analogs. A preferred target nucleic acid of the present inventionis RNA, especially ribosomal RNA (rRNA) and messenger RNA (mRNA).Ribosomal RNA is a preferred target nucleic acid for detecting groups oforganisms in test samples because of its relative abundance in cells andbecause of its conserved nature which allows for differentiating betweendefined groups of organisms. See, e.g., Kohne, “Method for Detecting,Identifying, and Quantitating Organisms and Viruses,” U.S. Pat. No.5,288,611, and Hogan et al., “Nucleic Acid Probes for Detection and/orQuantitation of Non-Viral Organisms,” U.S. Pat. No. 5,840,488. Formeasuring gene expression, determining the presence of a particularcell-type, or detecting the presence of a target group of viruses,assaying for specific mRNAs may be preferred. See, e.g., Gentalen etal., “Methods of Using an Array of Pooled Probes in Genetic Analysis,”U.S. Pat. No. 6,306,643; Kohne, “Method for Detecting the Presence ofRNA Belonging to an Organ or Tissue Cell-Type,” U.S. Pat. No. 5,932,416;and Kohne, “Method for Detecting the Presence of Group-Specific ViralmRNA in a Sample,” U.S. Pat. No. 5,955,261.

[0015] The sensitivity of an assay is limited by the amount of targetnucleic acid present in the test sample. To increase the sensitivity ofa detection assay, the present invention further contemplates anamplification step to increase the quantity of the target nucleic acidin the test sample prior to detection with a polynucleotide probe. Thus,the target nucleic acid may be directly obtained from the test sample orit may be a nucleic acid derived from an amplification procedure (i.e.,amplicon). Numerous amplification procedures are well known in the art,the most common of which is the polymerase chain reaction. See, e.g.,Mullis in U.S. Pat. No. 4,683,202. Amplification may be performed in thepresence of non-target nucleic acid or the target nucleic acid may beisolated and purified prior to amplification in order to removeinhibitors of amplification and to limit non-specific amplification.

[0016] The polycationic polymers and polynucleotide probes of thepresent invention may be provided to the test sample in any order. Thepolymers are synthetically produced and water soluble. They include aplurality of cationic charges and may be homopolymers and/or copolymers,including block and graft copolymers. While the polymers may includeionic and/or anionic monomers, the positively charged monomers arepresent in molar excess to the negatively charged monomers. The cationiccharges of the polymers may be localized or delocalized or they mayinclude both localized and delocalized cationic charges.

[0017] In a preferred embodiment, the polycationic polymers of thepresent invention form complexes of nanoparticle size in the test sampleunder hybridization assay conditions. These complexes may include aplurality of covalently linked polymers, and may further includecovalently linked polymers and polynucleotides. To prevent theirprecipitation out of solution, these complexes are preferably watersoluble. Thus, the cationic monomers present in the polymers arepreferably present in molar excess of the phosphate groups of the probesprovided to a test sample, and even more preferably present in molarexcess of the phosphate groups of all nucleic acids predicted to bepresent in a test sample.

[0018] In certain embodiments of the present invention, it is desirableto dissociate polymers and polynucleotides prior to detection of thetarget nucleic acid, if present, and after complementary polynucleotideshave had sufficient time to stably associate in the test sample.Dissociation can be achieved by providing one or more dissociatingagents to the test sample, such as an anionic detergent (e.g., lithiumlauryl sulfate) and/or a polyanion (e.g., exogenous nucleic acid). Thedissociating agents should be provided to the test sample in an amountsufficient to weaken the bonds between polymers and polynucleotides.

[0019] The association rate of complementary polynucleotides in thepresence of the polycationic polymers of the present invention ispreferably at least about 2-fold greater than the association rate ofthe same complementary polynucleotides in the absence of the polymerunder identical incubation periods and conditions. More preferably, theassociation rate is at least about 5-fold greater, at least about10-fold greater, at least about 100-fold greater or at least about1000-fold greater in the presence of the polycationic polymers. Suchconditions preferably include a temperature of at least about 40° C. anda salt concentration of at least about 5 mM monovalent cations (or anequivalent salt concentration including multivalent cations, such asdivalent magnesium present in magnesium chloride or magnesium sulfate).More preferably, such conditions include a temperature of at least about40° C. and a salt concentration of at least about 150 mM monovalentcations (or an equivalent salt concentration including multivalentcations). An equivalent salt concentration is one which will result inapproximately the same rate enhancement as a salt concentration whichdoes not include any multivalent cations under otherwise identicalconditions. The temperature of the reaction mixture will generally be inthe range of room temperature to about 90° C., and is preferably in therange of about 40° C. to about 70° C., more preferably in the range ofabout 50° C. to about 60° C., and is most preferably about 60° C.

[0020] In a further embodiment of the present invention, a kit isfeatured which comprises: (i) a polynucleotide probe whichpreferentially hybridizes to a target nucleic acid sequence in a testsample under hybridization assay conditions; and (ii) a syntheticpolycationic polymer in an amount sufficient to increase the associationrate of the probe and the target sequence in the test sample under thehybridization assay conditions. The probe and polymer may be provided inthe same or separate containers. Kits according to the present inventionmay further comprise at least one of the following: (i) a reagent todissociate the polymer from the probe; (ii) one or more amplificationprimers for amplifying a target sequence contained in or derived fromthe target nucleic acid; (iii) a capture probe for isolating andpurifying target nucleic acid present in a test sample; and (iv) if acapture probe is included, a solid support material (e.g., magneticallyresponsive particles) for immobilizing the capture probe, eitherdirectly or indirectly, in a test sample. Where the target nucleic acidis a structured nucleic acid having regions of self-complementarity,such as rRNA, the kits of the present invention may further includehelper probes. See Hogan et al., “Means and Method for Enhancing NucleicAcid Hybridization,” U.S. Pat. No. 5,030,557. Additionally, the kits maycomprise written instructions for performing an assay to determine thepresence or absence of a target nucleic acid sequence in the test sampleas an indication of the presence or absence of a target virus ororganism or members of a target group of viruses or organisms in thetest sample. The assay described in the written instructions may includesteps for isolating and purifying the target nucleic acid prior todetection with the polynucleotide probe and/or amplifying a targetsequence contained in the target nucleic acid. Alternatively, the kitmay include written instructions for performing, for example, an assayto detect the presence of a disease-associated gene, to determine thestate of a disease, to measure levels of gene expression, or to detectmutations or polymorphisms in a test sample.

[0021] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a plot of percent hybridization versus log of C_(o)t fora range of target concentrations used to determine the rate of areaction having a three minute incubation period, where each ()represents a data point derived from experimental data.

[0023]FIG. 2 is a plot of percent hybridization versus log of C_(o)t fora range of target concentrations used to determine the rate of areaction having an eight minute incubation period, where each ()represents a data point derived from experimental data.

[0024]FIG. 3 is a plot of the predicted percent hybridization versus logof C_(o)t curve (where the estimated association rate constant(k₁)=16,000 M⁻¹ and the estimated dissociation rate constant (k₂)=0)superimposed over the plotted data points () derived from experimentaldata and depicted in FIG. 1.

[0025]FIG. 4 is a plot of the predicted percent hybridization versus logof C_(o)t curve (where the estimated k₁=16,000 M⁻¹ and the estimatedk₂₌₀) superimposed over the plotted data points () derived fromexperimental data and depicted in FIG. 2.

[0026]FIG. 5 graphically represents an adjustment to k₁ and k₂(k₁=14,500 M⁻¹s⁻¹ and k₂=8.33×10⁻⁴s⁻¹), such that the predicted curve ofFIG. 3 and the data points () of FIG. 1 are coincident. Adjusted k₁ andk₂ should provide a closer approximation of the actual rate of thisreaction.

[0027]FIG. 6 graphically represents an adjustment to k₁ and k₂(k₁=14,500 M⁻¹s⁻¹ and k₂=8.33×10⁻⁴s⁻¹), such that the predicted curve ofFIG. 4 and the data points () of FIG. 2 are coincident. Adjusted k₁ andk₂ should provide a closer approximation of the actual rate of thisreaction.

[0028]FIG. 7 is a plot of percent hybridization versus log of C_(o)t fora range of target concentrations with polymer (♦) and without polymer(Δ), where each (♦) and (Δ) represents a data point derived fromexperimental data.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

[0029] The following terms have the indicated meanings in thespecification unless expressly indicated to have a different meaning.

[0030] By “target nucleic acid,” “target polynucleotide” or “target” ismeant a nucleic acid containing a target nucleic acid sequence.

[0031] By “target nucleic acid sequence,” “target sequence” or “targetregion” is meant a specific deoxyribonucleotide or ribonucleotidesequence comprising all or part of the nucleotide sequence of asingle-stranded nucleic acid molecule. Target nucleic acid sequences ofthe present invention contain a mixture of nucleotides, i.e., more thanone type of nucleotide (e.g., adenine (A), cytosine (T), guanine (G),inosine (I), thymine (T) or uracil (U)).

[0032] By “polynucleotide” is meant a polymer having two or morenucleoside subunits or nucleobase subunits coupled together. Thepolynucleotides include DNA and/or RNA or analogs thereof and mayfurther include non-nucleotide groups such as, for example, abasicnucleotides, universal bases (e.g., 3-nitropyrrole and 5-nitroindole),polysaccharides, peptides, polypeptides and/or polyethylene glycol. See,e.g., Becker et al., “Molecular Torches,” U.S. Pat. No. 6,361,945;Bergstrom et al., “3-Nitropyrrole Nucleoside,” U.S. Pat. No. 5,681,947;Loakes et al. Nucleic Acids Research (1995) 23(13):2361-2366; and Arnoldet al., “Linking Reagents for Nucleotide Probes,” U.S. Pat. No.5,585,481. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl substitution to the ribofuranosyl moiety.(Polynucleotides including nucleoside subunits having 2′ substitutionswhich are useful as polynucleotide probes are disclosed by Becker etal., “Method for Amplifying Target Nucleic Acid Using Modified Primers,”U.S. Pat. No. 6,130,038.) The nucleoside subunits may by joined bylinkages such as phosphodiester linkages, modified linkages or bynon-nucleotide moieties which do not prevent hybridization of thepolynucleotide to its complementary target nucleic acid sequence.Modified linkages include those linkages in which a standardphosphodiester linkage is replaced with a different linkage, such as aphosphorothioate linkage or a methylphosphonate linkage. The nucleobasesubunits may be joined, for example, by replacing at least a portion ofthe natural deoxyribose phosphate backbone of DNA with a pseuodo peptidebackbone, such as a 2-aminoethylglycine backbone which couples thenucleobase subunits by means of a carboxymethyl linker to the centralsecondary amine. (DNA analogs having a pseudo peptide backbone arecommonly referred to as “peptide nucleic acids” or “PNA” and aredisclosed by Nielsen et al. in U.S. Pat. No. 5,773,571.) Othernon-limiting examples of polynucleotides contemplated by the presentinvention include nucleic acid analogs containing bicyclic and tricyclicnucleoside and nucleotide analogs referred to as “Locked Nucleic Acids,”“Locked Nucleoside Analogues” or “LNA.” (Locked Nucleic Acids aredisclosed by Wang, “Conformationally Locked Nucleosides andOligonucleotides,” U.S. Pat. No. 6,083,482; U.S. Pat. No. 6,268,490; andWengel et al., “Oligonucleotide Analogues,” International PublicationNo. WO 99/14226.) Any nucleic acid analog is contemplated by the presentinvention provided the modified polynucleotide can form a stable hybridwith a target nucleic acid under hybridization assay conditions and atleast a portion of the modified polynucleotide is anionic. In the caseof polynucleotide probes, the modified polynucleotide must be capable ofpreferentially hybridizing to the target nucleic acid underhybridization assay conditions. Unless indicated to be a “probe,” apolynucleotide, as used herein, may be a nucleic acid molecule obtainedfrom a natural source which is at least partially single-stranded orwhich may be rendered partially or fully single-stranded by humanintervention.

[0033] By “polynucleotide probe” or “probe” is meant a polynucleotidehaving a base sequence sufficiently complementary to its target nucleicacid sequence to form a probe:target hybrid stable for detection underhybridization assay conditions. As would be understood by someone havingordinary skill in the art, a probe is an isolated nucleic acid molecule,or an analog thereof, in a form not found in nature without humanintervention (e.g., recombined with foreign nucleic acid, isolated, orpurified to some extent). Probes may have additional nucleosides ornucleobases outside of the targeted region so long as such nucleosidesor nucleobases do not prevent hybridization under hybridization assayconditions and, where indicated, do not prevent preferentialhybridization to the target nucleic acid. A non-complementary sequencemay also be included, such as a target capture sequence (generally ahomopolymer tract, such as a poly-A, poly-T or poly-U tail) or sequenceswhich will confer a desired secondary or tertiary structure, such as ahairpin structure, which can be used to facilitate detection. Examplesof probes having a target capture sequence (“capture probes”) aredisclosed by Ranki et al., “Detection of Microbial Nucleic Acids by aOne-Step Sandwich Hybridization Test,” U.S. Pat. No. 4,486,539;Stabinsky, “Methods and Kits for Performing Nucleic Acid HybridizationAssays,” U.S. Pat. No. 4,751,177; and Weisburg et al., “Two StepHybridization and Capture of a Polynucleotide,” U.S. Pat. No. 6,110,678.Self-hybridizing probes are disclosed by, for example, Bagwell,“Fluorescent Imperfect Nucleic Acid Probes,” U.S. Pat. No. 5,607,834;Tyagi et al., “Detectably Labeled Dual Conformation OligonucleotideProbes, Assays and Kits,” U.S. Pat. No. 5,925,517; and Becker et al. inU.S. Pat. No. 6,361,945.

[0034] Polynucleotide probes of the present invention are preferably nomore than about 100 bases in length, more preferably no more than about50 bases in length, and most preferably no more than about 35 bases inlength. Probes of a defined sequence may be produced by techniques knownto those of ordinary skill in the art, such as by chemical orbiochemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules, e.g., bacterial or retroviralvectors.

[0035] By “complementary” is meant polynucleotides having base sequenceregions able to form stable hydrogen bonds under hybridization assayconditions. Perfect complementarity between base regions ofpolynucleotides is not required, provided the two regions aresufficiently complementary to permit the stable formation of adouble-stranded, hydrogen-bonded region under hybridization assayconditions.

[0036] By “stably,” “stable” or “stable for detection” is meant that thetemperature of a reaction mixture is at least 2° C. below the meltingtemperature of a polynucleotide duplex. The temperature of the reactionmixture is more preferably at least 5° C. below the melting temperatureof the polynucleotide duplex, and even more preferably at least 10° C.below the melting temperature of the reaction mixture.

[0037] By “hybridization” is meant the ability of two completely orpartially complementary polynucleotides to come together under specifiedhybridization assay conditions in an orientation permitting theformation of a stable structure having a double-stranded region. The twoconstituent strands of this double-stranded structure, sometimes calleda hybrid, are held together by hydrogen bonds. Although these hydrogenbonds most commonly form between the bases of adenine and thymine oruracil (A and T or U) or cytosine and guanine (C and G) on singlenucleic acid strands, base pairing can also form between bases which arenot members of these “canonical” pairs. Non-canonical base pairing iswell-known in the art. (See, e.g., ROGER L. P. ADAMS ET AL., THEBIOCHEMISTRY OF THE NUCLEIC ACIDS (11^(th) ed. 1992).)

[0038] By “preferentially hybridize” is meant that under the specifiedhybridization assay conditions, polynucleotide probes can hybridize totheir target nucleic acids to form stable probe:target hybridsindicating the presence of a specific target nucleic acid sequence, andthere is not formed a sufficient number of stable probe:non-targethybrids to indicate the presence of non-target nucleic acids. Thus, theprobe hybridizes to target nucleic acid to a sufficiently greater extentthan to non-target nucleic acid to enable one having ordinary skill inthe art to accurately detect the presence (or absence) of the targetnucleic acid sequence in a test sample which may also contain non-targetnucleic acid. Probes which preferentially hybridize to target nucleicacid are particularly useful in diagnostic assays intended tospecifically detect the presence or absence of a particular virus ororganism or members of a group of viruses or organisms in a test samplewhich may also contain phylogenetically closely related non-targetviruses or organisms. Such diagnostic assays are well known in the artand are disclosed by, for example, Kohne in U.S. Pat. No. 5,288,611.

[0039] In general, reducing the degree of complementarity between apolynucleotide sequence and its target sequence will decrease the degreeor rate of hybridization of the polynucleotide to its target region.However, the inclusion of one or more non-complementary bases mayfacilitate the ability of a polynucleotide to discriminate againstnon-target polynucleotides.

[0040] Preferential hybridization can be measured using techniques wellknown to those having ordinary skill in the art, including thosedescribed in the Examples section infra. Preferably, there is at least a10-fold difference between target and non-target hybridization signalsin a test sample, more preferably at least a 100-fold difference, andmost preferably at least a 1,000-fold difference. Preferably, non-targethybridization signals in a test sample are no more than the backgroundsignal level.

[0041] By “phylogenetically closely related” is meant that the organismsor viruses are closely related to each other in an evolutionary senseand therefore would have a higher total nucleic acid sequence homologythan organisms or viruses that are more distantly related. Organisms orviruses occupying adjacent and next to adjacent positions on thephylogenetic tree are closely related. Organisms or viruses occupyingpositions farther away than adjacent or next to adjacent positions onthe phylogenetic tree will still be closely related if they havesignificant total nucleic acid sequence homology.

[0042] By “test sample” is meant a substance known or suspected tocontain target nucleic acid extracted or removed from any source,including bodily fluids, tissues, secretions and excretions, plants,water, food and the environment, for assaying in vitro or ex vivo. Thesubstance may be processed to isolate and purify nucleic acid containedtherein, such that use of the term “test sample” herein may refer toeither the substance in its unaltered, extracted state or to targetnucleic acid which has been isolated from the substance and thenpurified.

[0043] By “capture probe” is meant a polynucleotide or a set of at leasttwo polynucleotides linked together which are capable of hybridizing toa target nucleic acid and to an immobilized probe, thereby providingmeans for immobilizing and isolating the target nucleic acid in a testsample. That portion of the capture probe which hybridizes to the targetnucleic acid is referred to as the “target binding region,” and thatportion of the capture probe which hybridizes to the immobilized probeis referred to as the “immobilized probe binding region.” While thecapture probe hybridizes to both the target nucleic acid and theimmobilized probe under hybridization assay conditions, the targetbinding region and the immobilized probe binding region may be designedto hybridize to their respective target sequences under differenthybridization assay conditions. In this way, the capture probe may bedesigned so that it first hybridizes to the target nucleic acid undermore favorable in solution kinetics before adjusting the conditions topermit hybridization of the immobilized probe binding region to theimmobilized probe. The target binding and immobilized probe bindingregions may be directly adjoining each other on the same polynucleotide,they may be separated from each other by one or more optionally modifiednucleotides, and/or they may be joined to each other by means of anon-nucleotide linker.

[0044] By “target binding region” is meant that portion of apolynucleotide which stably binds to a target sequence present in atarget nucleic acid, a DNA or RNA equivalent of the target sequence or acomplement of the target sequence under hybridization assay conditions.The hybridization assay conditions may be stringent hybridization assayconditions or amplification conditions.

[0045] By “immobilized probe binding region” is meant that portion of apolynucleotide which hybridizes to an immobilized probe underhybridization assay conditions.

[0046] By “immobilized probe” is meant a polynucleotide for joining acapture probe to an immobilized support. The immobilized probe is joinedeither directly or indirectly to the solid support by a linkage orinteraction which remains stable under the conditions employed tohybridize the capture probe to the target nucleic acid and to theimmobilized probe, whether those conditions are the same or different.The immobilized probe facilitates separation of the bound target nucleicacid from unbound materials in a sample.

[0047] By “isolate,” “isolated” or “isolating” is meant that at least aportion of the target nucleic acid present in a test sample isconcentrated in a reaction receptacle or on a reaction device or solidcarrier (e.g., test tube, cuvette, microtiter plate well, nitrocellulosefilter, slide or pipette tip) in a fixed or releasable manner so thatthe target nucleic acid can be purified without significant loss of thetarget nucleic acid from the receptacle, device or carrier.

[0048] By “purify,” “purified” or “purifying” is meant that one or morecomponents of a sample present in a reaction receptacle or on a reactiondevice or solid carrier are physically removed from one or more othersample components present in the reaction receptacle or on the reactiondevice or solid carrier. Sample components which may be removed during aseparating or purifying step include proteins, carbohydrates, lipids,inhibitors, non-target nucleic acids and unbound probe. Preferablyretained in a sample during a purifying step are target nucleic acidsbound to immobilized capture probes.

[0049] By “reaction mixture” is meant a test sample containing apolynucleotide probe having a base region which is complementary to atarget sequence contained in a target nucleic acid known or suspected tobe present in the test sample. The reaction mixture is subjected toconditions which facilitate hybridization of the probe to the targetsequence.

[0050] By “hybridization conditions” or “hybridization assay conditions”is meant conditions permitting a polynucleotide probe to stablyhybridize to a target nucleic acid. Hybridization assay conditions mayvary depending upon factors including the GC (guanine/cytosine) contentand length of the probe, the degree of similarity between the probesequence and sequences of non-target nucleic acids which may be presentin the test sample, and the target sequence. Hybridization assayconditions include the temperature and composition of the hybridizationreagents or solutions. While the Examples section infra providespreferred hybridization assay conditions for detecting target nucleicacids, other acceptable conditions could be easily ascertained bysomeone having ordinary skill in the art.

[0051] By “reaction rate,” “rate of reaction,” “association rate” or“rate of association” is meant the rate at which polynucleotides havingregions of complementarity reassociate or hybridize to form duplexesunder hybridization assay conditions.

[0052] By “reassociation,” “reassociate,” “renaturation” or “renaturate”is meant the reformation of double-stranded polynucleotides fromsingle-stranded polynucleotides which were base-paired to each otherbefore being separated through a denaturation process.

[0053] By “hybridization” or “hybridize” is meant the formation ofdouble-stranded polynucleotides from single-stranded polynucleotides ofindividual origin with respect to each other (e.g., DNA from differentspecies or a mixture of RNA and DNA). As used herein, the term“hybridization” is used interchangeably to refer to hybridization orreassociation.

[0054] By “solution hybridization” or “in solution” is meant that thereactants present in a reaction mixture (i.e., polynucleotides havingcomplementary, single-stranded regions) are diffusible in the reactionmixture when they are exposed to hybridization assay conditions.

[0055] By “label” is meant a reporter moiety associated with apolynucleotide which can be detected by means well known in the art andused to indicate the presence or absence of a particular polynucleotidesequence in a test sample. Examples of labels which are well known inthe art include chemiluminescent, electrochemiluminescent andfluorescent compounds, radioisotopes, dyes, polynucleotides, enzymes,enzyme substrates, chromophores and haptens. When multiple interactinglabels are associated with a polynucleotide, interacting labels mayinclude, for example, the following: luminescent and quencher labels,luminescent and adduct labels, dye dimer labels, enzyme and substratelabels, enzyme and cofactor labels, and Forrester energy transfer pairs.Examples of polynucleotides having multiple interacting labels aredisclosed by, for example, Bagwell in U.S. Pat. No. 5,607,834; Tyagi etal. in U.S. Pat. No. 5,925,517; and Becker et al. in U.S. Pat. No.6,361,945.

[0056] By “nucleic acid duplex,” “duplex,” “nucleic acid hybrid” or“hybrid” is meant a stable nucleic acid structure comprising adouble-stranded, hydrogen-bonded region. Such hybrids include RNA:RNA,RNA:DNA and DNA:DNA duplex molecules and analogs thereof. The structureis sufficiently stable to be detectable by any known means, includingmeans which do not require a probe associated label. For instance, thedetection method may include a probe coated substrate which is opticallyactive and sensitive to changes in mass at its surface. Mass changesresult in different reflective and transmissive properties of theoptically active substrate in response to light and serve to indicatethe presence or amount of immobilized target nucleic acid. Thisexemplary form of optical detection is disclosed by Nygren et al.,“Devices and Methods for Optical Detection of Nucleic AcidHybridization,” U.S. Pat. No. 6,060,237. Other detection methods includethose based on detecting probe associated changes in conductivity orturbidity in the test sample.

[0057] By “helper probe” is meant a polynucleotide designed to hybridizeto a target nucleic acid at a different locus than that of apolynucleotide probe, thereby either increasing the rate ofhybridization of the probe to the target nucleic acid, increasing themelting temperature of the probe:target hybrid, or both.

[0058] By “amplification primer” or “primer” is meant a polynucleotidecapable of hybridizing to a target nucleic acid and acting as a primerand/or a promoter template (e.g., for synthesis of a complementarystrand, thereby forming a functional promoter sequence) for theinitiation of nucleic acid synthesis. If the amplification primer isdesigned to initiate RNA synthesis, the primer may contain a basesequence which is non-complementary to the target sequence but which isrecognized by an RNA polymerase, such as a T7, T3 or SP6 RNA polymerase.An amplification primer may contain a 3′ terminus which is modified toprevent or lessen the rate or amount of primer extension. (McDonough etal. disclose primers and promoter-primers having modified or blocked3′-ends in U.S. Pat. No. 5,766,849, entitled “Methods of AmplifyingNucleic Acids Using Promoter-Containing Primer Sequences.”) While theamplification primers of the present invention may be chemicallysynthesized or derived from a vector, they are not naturally-occurringnucleic acid molecules.

[0059] By “nucleic acid amplification,” “target amplification” or“amplification” is meant increasing the number of nucleic acid moleculeshaving at least one target nucleic acid sequence. Target amplificationaccording to the present invention may be either linear or exponential,although exponential amplification is preferred.

[0060] By “amplicon” is meant a nucleic acid molecule generated in anucleic acid amplification reaction and which is derived from a targetnucleic acid. An amplicon contains a target nucleic acid sequence whichmay be of the same or opposite sense as the target nucleic acid.

[0061] By “derived” is meant that the referred to nucleic acid isobtained directly from a target organism or indirectly as the product ofa nucleic acid amplification, which product may be, for instance, anantisense RNA molecule which does not exist in the target organism.

[0062] By “polymer” is meant a macromolecule comprising one or morerepeated monomer types of low relative molecular mass covalently joinedtogether.

[0063] By “polycationic polymer” is meant a polymer having a netpositive charge, whether the charges of the polymer are localized ordelocalized. A polycationic polymer is comprised of at least onecationic monomer type and may also include anionic and/or nonionicmonomer types, provided the polymer has a net positive charge.

[0064] By “monomer” is meant a molecule which can undergopolymerization, thereby contributing constitutional units to theessential structure of a polymer.

[0065] By “constitutional units” is meant an atom or group of atoms(including pendant atoms or groups, if any) comprising a part of theessential structure of a polymer.

[0066] By “block copolymer” is meant a polymer composed of blocks inlinear sequence.

[0067] By “block” is meant a portion of a polymer, comprising manyconstitutional units, which has at least one feature that is not presentin adjacent portions of the polymer.

[0068] By “graft copolymer” is meant a polymer having one or morespecies of block connected to the main chain of the polymer as sidechains. These side chains have constitutional or configurationalfeatures that differ from those of the main chain.

[0069] By “synthetic” is meant that the polymerization of the polymerdid not occur exclusively in nature without human intervention.

[0070] By “complex” is meant a composition comprising a plurality ofpolycationic polymers. Complexes of the present invention are generallynanoparticles.

[0071] By “degree of polymerization” or “DP” is meant the number ofrepeating units in a polymer chain.

[0072] By “number-average molecular weight” or “M_(n)” is meant a valueequal to the weight of a polymer mixture divided by the number ofmolecules in the mixture. The number-average molecular weight can bedetermined by various well known methods, including colligativeproperties, osmotic pressure and freezing point depression methods.

[0073] By “weight-average molecular weight” or “M_(w)” is meant theaverage molecular weight of the molecules in a polymer mixture. Theweight-average molecular weight can be determined by a number ofdifferent well known methods, including light-scattering andultracentrifuge methods. While the M_(w) value and the M_(n) value maybe the same if all of the molecules in a mixture have essentially thesame weight, the M_(w) value will be higher than the M_(n) value if someof the molecules in the mixture are heavier than others.

[0074] By “polydispersity” is meant the ratio of the weight-averagemolecular weight and number-average molecular weight (M_(w)/M_(n)) in apolymer mixture. The polydispersity value indicates how wide the rangeof molecular weights is in a mixture.

[0075] The following abbreviations have the indicated meanings:“Da”=daltons; “M”=moles/liter; “s”=seconds; and “m”=minutes.

B. Hybridization Conditions and Probe Design

[0076] Hybridization reaction conditions, most importantly thetemperature of hybridization and the concentration of salt in thehybridization solution, can be selected to allow a polynucleotide probeto preferentially hybridize to a target nucleic acid and not tonon-target nucleic acid known or suspected of being present in a testsample. At decreased salt concentrations and/or increased temperatures(conditions of increased stringency) the extent of hybridizationdecreases as hydrogen bonding between paired nucleotide bases in thedouble-stranded hybrid molecule is disrupted. This process is known as“melting.”

[0077] Generally speaking, the most stable hybrids are those having thelargest number of contiguous, perfectly matched (i.e., hydrogen-bonded)nucleotide base pairs. Such hybrids would usually be expected to be thelast to melt as the stringency of the hybridization conditionsincreases. However, a double-stranded nucleic acid region containing oneor more mismatched, “non-canonical,” or imperfect base pairs (resultingin weaker or non-existent base pairing at that position in thenucleotide sequence of a nucleic acid) may still be sufficiently stableunder conditions of relatively high stringency to allow the nucleic acidhybrid to be formed and detected in a hybridization assay withoutcross-reacting with other, non-selected nucleic acids which may bepresent in a test sample.

[0078] Hence, depending on the degree of similarity between thenucleotide sequences of the target nucleic acid and those of non-targetnucleic acids present in the test sample on one hand, and the degree ofcomplementarity between the base sequence of a particular probe and thenucleotide sequences of the target and non-target nucleic acids on theother, one or more mismatches will not necessarily defeat the ability ofa probe to hybridize to the target nucleic acid and not to non-targetnucleic acids present in the test sample.

[0079] Polynucleotide probes useful in the present invention arepreferably chosen, selected, and/or designed to maximize the differencebetween the melting temperatures T_(m) of the probe:target hybrid andthe T_(m) of a mismatched hybrid formed between the probe and non-targetnucleic acid present in the test sample (e.g., nucleic acid, such asribosomal RNA (rRNA), from a phylogenetically closely-related organism).

[0080] Where the target nucleic acid is rRNA, it is important to notethat within the rRNA molecule there is a close relationship betweensecondary structure (caused in part by intra-molecular hydrogen bonding)and function. This fact imposes restrictions on evolutionary changes inthe primary nucleotide sequence causing the secondary structure to bemaintained. For example, if a base is changed in one “strand” of adouble helix (due to intra-molecular hydrogen bonding, both “strands”are part of the same rRNA molecule), a compensating substitution usuallyoccurs in the primary sequence of the other “strand” in order topreserve complementarity (this is referred to as co-variance), and thusthe necessary secondary structure. This allows two very different rRNAsequences to be aligned based both on the conserved primary sequence andalso on the conserved secondary structure elements. Potential targetsequences for the polynucleotide probes can be identified by notingvariations in the homology of the aligned sequences.

[0081] Merely identifying a putatively unique potential targetnucleotide sequence does not guarantee that a functionally specificpolynucleotide probe may be made to hybridize to nucleic acid comprisingthat sequence. Various other factors will determine the suitability of anucleic acid locus as a target site for a specific probe. Because theextent and specificity of hybridization reactions, such as thosedescribed herein, are affected by a number of factors, manipulation ofone or more of those factors will determine the exact sensitivity andspecificity of a particular polynucleotide, whether perfectlycomplementary to its target or not. The importance and effect of varioushybridization assay conditions are known to those skilled in the art andare disclosed by, for example, Kohne, “Method for Detection,Identification and Quantitation of Non-Viral Organisms,” U.S. Pat. No.4,851,330; Hogan et al., “Nucleic Acid Probes to Mycobacteriumgordonae,” U.S. Pat. No. 5,216,143; and Hogan, “Nucleic Acid Probes forDetection and/or Quantitation of Non-Viral Organisms,” U.S. Pat. No.5,840,488.

[0082] The desired temperature of hybridization and the hybridizationsolution composition (such as salt concentration, detergents and othersolutes) can also greatly affect the stability of double-strandedhybrids. Conditions such as ionic strength and the temperature at whicha probe will be allowed to hybridize to a target must be taken intoaccount in constructing a probe. As noted above, the thermal stabilityof hybrid polynuceotides generally increases with the ionic strength ofthe reaction mixture. On the other hand, chemical reagents which disrupthydrogen bonds, such as formamide, urea, dimethyl sulfoxide andalcohols, can greatly reduce the thermal stability of the hybrids.

[0083] To maximize the specificity of a probe for its target, probesshould be designed to hybridize to their targets under conditions ofhigh stringency. Under such conditions only polynucleotides (or regions)having a high degree of complementarity will hybridize to each other.Polynucleotides without such a high degree of complementarity will notform hybrids. Accordingly, the stringency of the hybridization assayconditions determines the amount of complementarity which should existbetween two polynucleotides in order to form a hybrid. Stringency ischosen to maximize the difference in stability between the hybrid formedbetween the probe and the target nucleic acid and potential hybridsbetween the probe and any non-target nucleic acids present in a testsample.

[0084] While probes having extensive self-complementarity are generallyavoided, there are some applications in which probes exhibiting at leastsome degree of self-complementarity are desirable to facilitatedetection of probe:target duplexes in the presence of unhybridizedprobe. By way of example, structures referred to as “Molecular Torches”are designed to include distinct regions of self-complementarity (coinedthe “target binding domain” and the “target closing domain”) which areconnected by a polynucleotide and/or non-nucleotide joining region andhybridize to one another under predetermined hybridization assayconditions. When exposed to denaturing conditions, the two complementaryregions (which may be fully or partially complementary) of the MolecularTorch melt, leaving the target binding domain available forhybridization to a target sequence when the predetermined hybridizationassay conditions are restored. Molecular Torches are designed so thatthe target binding domain favors hybridization to the target sequenceover the target closing domain. The target binding domain and the targetclosing domain of a Molecular Torch include interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the Molecular Torch is self-hybridized than when the MolecularTorch is hybridized to a target nucleic acid, thereby permittingdetection of probe:target duplexes in a test sample in the presence ofunhybridized probe having viable labels associated therewith. (MolecularTorches are disclosed by Becker et al. in U.S. Pat. No. 6,361,945.) Inaccordance with the teachings of Becker, probes of the present inventionmay be designed and constructed to include, in addition to a “targetbinding domain” able to distinguish target nucleic acid from non-targetnucleic acid, a “target closing domain,” a “joining region” andinteracting labels characteristic of a Molecular Torch.

[0085] Another example of a self-complementary probe is a structureknown as a “Molecular Beacon.” Molecular Beacons include nucleic acidmolecules having a target complement sequence, an affinity pair (ornucleic acid arms) holding the probe in a closed conformation in theabsence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe target nucleic acid and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). (Molecular Beaconsare disclosed by Tyagi et al. in U.S. Pat. No. 5,925,517.) In accordancewith the teachings of Tyagi, probes of the present invention may bedesigned and constructed to include, in addition to a “target complementsequence” able to distinguish target nucleic acid from non-targetnucleic acid, an “affinity pair” and dual labels characteristic of aMolecular Beacon.

[0086] Specificity may be achieved by limiting that portion of thepolynucleotide probe having perfect complementarity to non-targetsequences, by avoiding G and C rich regions of complementarity tonon-target nucleic acids, and by constructing the probe to contain asmany destabilizing mismatches to non-target sequences as possible.Whether a probe is appropriate for detecting a specific target nucleicacid depends largely on the thermal stability difference betweenprobe:target hybrids versus probe:non-target hybrids. In designingprobes, the differences in these T_(m) values should be as large aspossible (preferably 2° to 5° C. or more). Manipulation of the T_(m) canbe accomplished by changes to probe length and probe composition, suchas GC content versus AT content or the inclusion of nucleotide analogs(e.g., ribonucleotides having a 2′-O-methyl substitution to theribofuranosyl moiety).

[0087] In general, the optimal hybridization temperature forpolynucleotide probes is approximately 5° C. below the meltingtemperature for a given duplex. Incubation at temperatures below theoptimum temperature may allow mismatched base sequences to hybridize andcan therefore decrease specificity. The longer the probe, the morehydrogen bonding between base pairs and, in general, the higher theT_(m). Increasing the percentage of G and C also increases the T_(m)because G-C base pairs exhibit additional hydrogen bonding and thereforegreater thermal stability than A-T base pairs. Such considerations arewell known in the art. See, e.g., J. SAMBROOK ET AL., MOLECULAR CLONING:A LABORATORY MANUAL, ch. 11 (2d ed. 1989).

[0088] A preferred method for determining T_(m) measures hybridizationusing the Hybridization Protection Assay (HPA) disclosed by Arnold etal., “Homogenous Protection Assay,” U.S. Pat. No. 5,283,174. The T_(m)can be measured using HPA in the following manner. Probe molecules arelabeled with an acridinium ester and permitted to form probe:targethybrids in a lithium succinate buffer (0.1 M lithium succinate buffer,pH 4.7, 20 mM EDTA, 15 mM aldrithiol-2, 1.2 M LiCl, 3% (v/v) ethanolabsolute, 2% (w/v) lithium lauryl sulfate) using an excess amount oftarget. Aliquots of the solution containing the probe:target hybrids arethen diluted in the lithium succinate buffered solution and incubatedfor five minutes at various temperatures starting below that of theanticipated T_(m) (typically 55° C.) and increasing in 2-5° C.increments. This solution is then diluted with a mild alkaline boratebuffer (600 mM boric acid, 240 mM NaOH, 1% (v/v) TRITON® X-100, pH 8.5)and incubated at an equal or lower temperature (for example 50° C.) forten minutes.

[0089] Under these conditions the acridinium ester attached to thesingle-stranded probe is hydrolyzed, while the acridinium ester attachedto hybridized probe is relatively protected from hydrolysis. Thus, theamount of acridinium ester remaining after hydrolysis treatment isproportional to the number of hybrid molecules. The remaining acridiniumester can be measured by monitoring the chemiluminescence produced fromthe remaining acridinium ester by adding hydrogen peroxide and alkali tothe solution. Chemiluminescence can be measured in a luminometer, suchas a LEADER® 450i luminometer (Gen-Probe Incorporated, San Diego,Calif.; Cat. No. 3200i). The resulting data is plotted as percent ofmaximum signal (usually from the lowest temperature) versus temperature.The T_(m) is defined as the temperature at which 50% of the maximumsignal remains. In addition to the method above, T_(m) may be determinedby isotopic methods known to those skilled in the art, such as thosedisclosed by Hogan in U.S. Pat. No. 5,840,488.

[0090] To ensure specificity of a probe for its target, it is preferableto design probes which hybridize only to target nucleic acid underconditions of high stringency. Only highly complementary sequences willform hybrids under conditions of high stringency. Accordingly, thestringency of the hybridization assay conditions determines the amountof complementarity needed between two sequences in order for a stablehybrid to form. Stringency should be chosen to maximize the differencein stability between the probe:target hybrid and potentialprobe:non-target hybrids. See SAMBROOK ET AL., supra, ch. 11.

[0091] The length of the target nucleic acid sequence region and,accordingly, the length of the probe sequence can also be important. Insome cases, there may be several sequences from a particular region,varying in location and length, which may be used to design probes withthe desired hybridization characteristics. In other cases, one probe maybe significantly better with regard to specificity than another whichdiffers from it merely by a single base. While it is possible forpolynucleotides that are not perfectly complementary to hybridize, thelongest stretch of perfectly complementary bases, as well as the basecompositions, will generally determine hybrid stability.

[0092] If a target nucleic acid is wholly or partially involved in anintra-molecular or inter-molecular hybrid, it will be less able toparticipate in the formation of a new inter-molecular probe:targethybrid without a change in the reaction conditions. Ribosomal RNAmolecules are known to form very stable intra-molecular helices andsecondary structures by hydrogen bonding. By designing a probe to aregion of the target nucleic acid which remains substantiallysingle-stranded under hybridization assay conditions, the rate andextent of hybridization between probe and target may be increased.However, if the preferred target region is contained in region of anrRNA molecule which is at least partially double-stranded, then helperprobes may used to facilitate access to the target region. A helperprobe is a polynucleotide which is designed to hybridize to the targetnucleic acid at a different locus than that of a polynucleotide probe,thereby increasing the rate of hybridization of the polynucleotide probeto the target nucleic acid, increasing the melting temperature of theprobe:target hybrid, or both. Hogan et al. disclose helper probes inU.S. Pat. No. 5,030,557.

[0093] A genomic target occurs naturally in a double-stranded form, asdoes a product of the polymerase chain reaction (PCR) method ofamplification. These double-stranded targets are naturally inhibitory tohybridization with a probe and require denaturation prior tohybridization. Appropriate denaturation and hybridization conditions areknown in the art. See, e.g., Southern J. Mol. Biol. (1975) 98:503.

[0094] A number of formulae are available which will provide an estimateof the melting temperature for perfectly matched polynucleotides totheir target nucleic acids. One such formula is the following:

T _(m)=81.5+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−(600/N)

[0095] (where N=the length of the polynucleotide in number ofnucleotides) provides a good estimate of the T_(m) for polynucleotidesbetween 14 and 60 to 70 nucleotides in length. From such calculations,subsequent empirical verification or “fine tuning” of the T_(m) may bemade using screening techniques well known in the art. For furtherinformation on hybridization and polynucleotide probes, reference may bemade to SAMBROOK ET AL., supra, ch. 11. This reference, among otherswell known in the art, also provides estimates of the effect ofmismatches on the T_(m) of a hybrid. Thus, from known nucleotidesequences, polynucleotides may be designed which can distinguish betweenthese sequences.

[0096] C. Preparation of Polynucleotide Probes

[0097] The polynucleotide probes used in the present invention can bereadily prepared by methods known in the art. Preferably, the probes aresynthesized using solid phase methods. For example, Caruthers describesusing standard phosphoramidite solid-phase chemistry to join nucleotidesby phosphodiester linkages. See Caruthers et al. Methods Enzymol. (1987)154:287. Automated solid-phase chemical synthesis using cyanoethylphosphoramidite precursors has been described by Barone. See Barone etal. Nucleic Acids Res. (1984) 12(10):4051. Likewise, a procedure forsynthesizing polynucleotides containing phosphorothioate linkages isdisclosed by Batt, “Method and Reagent for Sulfurization ofOrganophosphorous Compounds,” U.S. Pat. No. 5,449,769. In addition, thesynthesis of polynucleotides having different linkages includingmethylphosphonate linkages are disclosed by Riley et al., “Process forthe Purification of Oligomers,” U.S. Pat. No. 5,811,538. Moreover,methods for the organic synthesis of polynucleotides are known to thoseof skill in the art and are disclosed by, for example, SAMBROOK ET AL.,supra, ch. 11.

[0098] Following synthesis and purification of a particularpolynucleotide, several different procedures may be utilized to purifyand control the quality of the polynucleotide. Suitable proceduresinclude polyacrylamide gel electrophoresis or high pressure liquidchromatography. Both of these procedures are well known to those skilledin the art.

[0099] Polynucleotides which can be used in the present invention may bemodified with chemical groups to enhance their performance, providedthose polynucleotides being used as probes carry a net positive charge.For example, backbone-modified polynucleotides, such as those havingphosphorothioate, methylphosphonate, 2′-O-alkyl or peptide groups whichrender the polynucleotides resistant to the nucleolytic activity ofcertain polymerases or to nuclease enzymes may allow the use of suchenzymes in an amplification or other reaction. Another example of amodification involves using non-nucleotide linkers incorporated betweennucleotides in the nucleic acid chain of a polynucleotide, and which donot prevent hybridization of a probe. See Arnold et al., “Non-NucleotideLinking Reagents for Nucleotide Probes,” U.S. Pat. No. 6,031,091. Thepolynucleotides of the present invention may also contain mixtures ofthe desired modified and natural nucleotides.

[0100] Once synthesized, a selected polynucleotide may be labeled by anyof several well known methods (see, e.g., SAMBROOK ET AL., supra, ch.10). Useful labels include radioisotopes as well as non-radioactivereporting groups. Isotopic labels include ³H, ³⁵S, ³²P, 125i, ⁵⁷Co and¹⁴C. Isotopic labels can be introduced into the polynucleotide bytechniques known in the art such as nick translation, end labeling,second strand synthesis, the use of reverse transcription, and bychemical methods. When using radiolabeled probes, hybridization can bedetected by autoradiography, scintillation counting or gamma counting.The detection method selected will depend upon the particularradioisotope used for labeling.

[0101] Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups, as disclosed by Arnold et al.in U.S. Pat. No. 6,031,091. Non-isotopic labels include fluorescentmolecules (individual labels or combinations of interacting labels, suchas the fluorescence resonance energy transfer (FRET) pairs disclosed byTyagi et al. in U.S. Pat. No. 5,925,517), chemiluminescent molecules,enzymes, cofactors, enzyme substrates, haptens or other ligands. Theprobes of the present invention are preferably labeled by means of anon-nucleotide linker with an acridinium ester (AE). Acridinium esterlabeling may be performed as disclosed by Arnold et al., “AcridiniumEster Labelling and Purification of Nucleotide Probes,” U.S. Pat. No.5,185,439.

D. Polycationic Polymers

[0102] Polymers of the present invention have a net cationic charge andinclude polymers of one repetitive monomer type (i.e., homopolymers) aswell as polymers of multiple repetitive monomer types (i.e.,copolymers). Polymers of the present invention further include block andgraft copolymers. Those polymers having multiple monomer types mayinclude, in addition to cationic monomers, monomers which are ionic oranionic or both, provided the cationic charges of the polymers are inexcess of the anionic charges. The cationic charges of the polymers maybe localized or delocalized (i.e., localized to a particular monomer orspread over two or more contiguous monomers). In one preferredembodiment, the distance between adjacent cationic monomers of thepolymers approximates the distance between adjacent phosphate groups ofa polynucleotide, which is in the range of about 5 to about 7 angstroms.The polymers of the present invention are synthetic and water soluble.

[0103] The polycationic polymers of the present invention promote thereassociation or hybridization (collectively referred to herein as“association”) of complementary polynucleotides by greatly increasingthe rate at which polynucleotides associate in solution, even inreaction mixtures containing medium to high salt concentrations (i.e.,salt concentrations greater than about 150 mM for monovalent cations,such as lithium (Li+), potassium (K+) and sodium (Na+)). Reactionmixtures having salt concentrations greater than about 5 to about 10 mMfor monovalent cations are preferred in the present invention. The rateenhancement due to the presence of polycationic polymers in a reactionmixture may be as great as 2-fold, 5-fold, 10-fold, 100-fold, 1000-foldor greater. Determining the effect that any particular polycationicpolymer or group of polycationic polymers has on the rate of associationof complementary polynucleotides can be evaluated using referenceconditions that differ only by the presence or absence of thepolycationic polymer or group of polycationic polymers. The referenceconditions used herein included either a high salt hybridization buffer(100 mM lithium succinate, pH 5.1, 0.35 M LiCl and 0.1% (v/v) TRITON®X-100) or a low salt hybridization buffer (100 mM lithium succinate, pH5.1, 50 mM LiCl and 0.1% (v/v) TRITON® X-100) and an incubationtemperature of 40° C. or 60° C. The reference conditions may be anyconditions which facilitate stable hybridization between complementarypolynucleotides. Other conditions of stringency could serve as referenceconditions for determining the effect of a polycationic polymer or agroup of polycationic polymers on the rate of association betweencomplementary polynucleotides, including the standard reactionconditions of 0.18 M Na⁺ (0.12 M sodium phosphate buffer, pH 6.8) at 60°C. See Britten et al. Methods Enzymol. (1974) 29:363-418.

[0104] Polycationic polymers are known to significantly increase theT_(m) of nucleic acid duplexes, and large increases in T_(m) are oftenassociated with a loss in discrimination. See, e.g., Maruyama et al.Bioconjugate Chem. (1998) 9:292-299 and Majlessi et al. Nucleic AcidsResearch (1998) 26:2224-2229. For detection assays, in whichpolynucleotide probes are intended to preferentially hybridize to targetnucleic acid in the presence of non-target nucleic acid, the probes mustbe specific for the target nucleic acid in order for the assay to be ofdiagnostic or probative value. A loss in discrimination cannot betolerated. Thus, the applicant's discovery that polycationic polymers ofthe present invention can be used in an assay employing generallypracticed salt and temperature conditions to significantly increase theT_(m) of hybrids, without a loss in specificity for the target nucleicacid, was unexpected. The polycationic polymers of the present inventionappear to function by further stabilizing the small duplex formed duringnucleation, but not to such an extent that the probe loses itsspecificity for the target nucleic acid. (Nucleation sites have beendescribed as spanning as few as three adjacent base pairs, and may be aslong as six to eight adjacent base pairs. See Porschke et al. J. Mol.Biol. (1971) 62:361-381.) As a result, the applicant surprisingly foundthat the polycationic polymers of the present invention can be used toincrease the reaction rate of assays in which the probe preferentiallyhybridizes to a target nucleic acid in the presence of non-targetnucleic acid, even nucleic acid from a closely related non-targetorganism or virus.

[0105] Through routine screening, the applicant also discovered thatpolycationic polymers can be selected which allow for some mismatchtolerance so that closely related strains of an organism or virus, forexample, can be detected in an assay. These polymers would allowpractitioners to design probes which are less sensitive to sequencevariation so that multiple strains of an organism or virus could bedetected with a single probe, while still having sufficient specificityto distinguish over non-target nucleic acid present in a test sample.Assays employing these selected for polymers would be particularlyuseful in detecting organisms or viruses exhibiting high genotypicdiversity, such as the HIV-1 and HIV-2 viruses. See, e.g., Chee et al.,“Array of Nucleic Acid Probes on Biological Chips for Diagnosis of HIVand Methods of Using the Same,” U.S. Pat. No. 5,861,242. By adjustingthe conditions of stringency (e.g., salt and temperature conditions), itis expected that these same polymers could be used in assays requiringsingle base mismatch discrimination.

[0106] While not wishing to be bound by theory, the applicant currentlybelieves that the polycationic polymers of the present inventionassemble in solution to form complexes of nanometer dimensions (i.e.,nanoparticles) which create a charge environment that attractsnegatively charged polynucleotides (e.g., polynucleotide probes andtarget nucleic acids) present in the solution. Once localized by thesecomplexes, polynucleotides having sufficiently complementary sequencesare able to more readily associate, thereby enhancing the associationkinetics of complementary polynucleotides. Alternatively, the applicanttheorizes that the polycationic polymers of the present inventionassemble in solution as they bind polynucleotides, such that thepolynucleotides are being localized as the nanoparticles are formingrather than after the nanoparticles have already largely formed.Microscopy methods well known to those skilled in the art could be usedto screen for the formation of nanoparticles comprising polycationicpolymers.

[0107] Based upon the applicant's theory that the formation of complexesattracts and concentrates polynucleotides present in a reaction mixture,it is preferable that the molar concentration of cationic monomers whichare present in the polycationic polymers of the reaction mixture exceedthe molar concentration of anionic phosphate groups (i.e., nucleotides)which are present in the polynucleotides of the reaction mixture.Providing a molar excess of cationic monomers to a reaction mixtureshould also prevent complexes of polycationic polymers fromprecipitating out of solution, as may occur when approximately equalnumbers of cationic monomers and phosphate groups are provided to thereaction mixture. Precipitation is undesirable since nucleic acids areremoved from solution, thereby impeding hybridization betweencomplementary polynucleotides. While even small amounts of thepolycationic polymers should enhance the rate of association in areaction mixture, preferred concentrations of the polycationic polymersare in the range of about 1 μM to about 1000 μM, and more preferably inthe range of about 10 μM to about 100 μM.

[0108] The weight average molecular weights (M_(w)) of polycationicpolymers of the present invention are preferably less than about 300,000Da, as polymers having a M_(w) greater than about 300,000 Da are oftentoo viscous or polydisperse to adequately facilitate the association ofcomplementary polynucleotides. While the lowest acceptable M_(w) willdepend greatly upon the polymer used, polymers having a M_(w) of atleast 10,000 Da are generally preferred. Polymers having optimal M_(w)values for enhancing the association kinetics of complementarypolynucleotides can be determined through routine screening proceduresfor any given set of reaction conditions.

[0109] Although the polycationic polymers of the present invention maybe polydisperse, it is generally preferred that the polymers of amixture have a polydispersity value of about one. Since polydispersityis a measure of the ratio of the weight-average molecular weight andnumber-average molecular weight (M_(w)/M_(n)) in a polymer mixture, thecloser this ratio is to one, the greater the size uniformity of thepolymers making up the mixture.

[0110] Polycationic polymers contemplated by the present inventioninclude, but are not limited to, poly-L-lysine (an example of which ispoly-L-lysine hydrobromide available from Fluka AG of Buchs, Switzerlandas Cat. Nos. 81333 and 81355), poly(lys, tyr) 4:1 (an example of whichis poly(lys, tyr) 4:1 hydrobromide available from Sigma Chemical Companyof St. Louis, Mo. as Product No. P 4659), poly-L-histidine (an exampleof which is poly-L-histidine hydrochloride available from Sigma ChemicalCompany as Product No. P 2534), poly-L-arginine (an example of which ispoly-L-arginine hydrochloride available from Sigma Chemical Company asProduct No. P 4663), hexadimethrine bromide(1,5-dimethyl-1,5-diazaundeca-methylene polymethobromide) (an example ofwhich is available from Sigma Chemical Company as Product No. H 9268),poly(allylamine hydrochloride) (an example of which is available fromAldrich Chemical Company of Milwaukee, Wis. as Cat. No. 28,321-5),poly(diallyldimethylammonium chloride) (an example of which is availablefrom Aldrich Chemical Company as Cat. No.40,901-4),poly[bis(2-chloroethyl)ether-alt-1,3 bis[3-(dimethyl amino) propyl]urea], polyethylenimine (an example of which is available from AldrichChemical Company as Cat. No. 40,872-7) and poly-L-lysine dextran. Whilenot specifically enumerated, other polycationic polymers are envisagedby the present invention which function to enhance the associationkinetics of complementary polynucleotides. Such polycationic polymerscan be easily screened for by skilled artisans following the guidanceprovided herein without having to engage in anything more than routineexperimentation.

[0111] The polycationic polymers of the present invention may be used inconjunction with other techniques for increasing reaction rates,including techniques which depend upon volume exclusion to enhance therate of association between complementary polynucleotides. By addingsynthetic polymers such as polyetheylene glycol, dextran or dextransulfate, the volume of a reaction mixture which remains available to thepolynucleotide reactants is reduced, thereby increasing the effectiveconcentration of these reactants. Volume exclusion techniques are wellknown in the art and are described in, for example, Renz et al. NucleicAcids Res. (1984) 12:3435-3444 and Wahl et al. Proc. Natl. Acad. Sci.USA (1979) 75:3683-3687. Other techniques for increasing the rate ofassociation between polynucleotides which may be used in combinationwith the polymers of the present invention could include techniqueswhich enhance reaction rates by including a precipitating agent. See,e.g., Kohne et al., “Accelerated Nucleic Acid Reassociation Method,”U.S. Pat. No. 5,132,207.

E. Association Kinetics

[0112] The term “association rate,” as used herein, refers to the rateat which two polynucleotides reassociate or hybridize to form a duplexin solution. A number of factors affect the association rate, includingthe size and concentration of the polynucleotides, the incubationtemperature and the salt concentration of the reaction mixture.Conventionally, association rates have been quantified using the C_(o)tanalysis developed by Britten and Kohne. Science (1968) 161:529-540.Following this analysis, the fraction of single-stranded polynucleotidesremaining at any time during an isothermal reaction is given by theformula: C/C_(o)=1/(1+kC_(o)t), where C_(o) is the startingconcentration of the polynucleotides in nucleotides per liter, C is theconcentration of the polynucleotides in nucleotides per liter remainingat any given time, t is the time of the reaction (s), and k is the rateconstant for a second-order reaction (M⁻¹s⁻¹). When a reaction is halfcomplete (time=t_(1/2)), C/C_(o)=½ and C_(o)t_(1/2)=1/k. Thus,C_(o)t_(1/2) is inversely proportional to the rate constant and is ameasure of the association rate.

[0113] A shortcoming of the C_(o)t analysis method for measuringassociation rates is that it does not account for the simultaneousdissociation of polynucleotides during a reaction. The amount ofdissociation that takes place during a reaction will depend on suchfactors as the temperature of the reaction, as it relates to the meltingtemperature of the duplex formed between polynucleotides, and the timeof the reaction. As the reaction temperature approaches the meltingtemperature of the duplex or as the reaction period increases, theamount of dissociation is expected to rise. Therefore, to accuratelycalculate the rate of association, it is necessary to factor in both theassociation rate constant and the dissociation rate constant of thereaction.

[0114] To make this determination, the applicant devised a novelequation and method for calculating a rate constant for measuring therate of a reaction which accounts for both association and dissociationof the involved polynucleotides. The first step in this procedure is toplot percent hybridization versus log of C_(o)t data points on a graphfor each of the polynucleotide concentrations tested. Examples of suchplots are depicted in FIGS. 1 and 2, which plot data points ()calculated from the experimental data of Example 1 infra. These graphsplot data points for hybridizations of both 3 and 8 minutes. Twoincubation periods are used to ensure that the association ofpolynucleotides is increasing with time so that it can be establishedthat the rate being calculated is a kinetic determination and not anequilibrium measurement. An increase in the percentage of hybridizationcan be demonstrated by showing that the percentage of hybridization isincreasing with both concentration and time.

[0115] Next, predicted curves are plotted based on percent hybridizationvalues calculated from the following novel equation: %hybridization=100(1−K)(1−exp−(k₁C_(o)+k₂)t), where K=k₂/(C_(o)k₁+k₂),t=time (s), C_(o)=initial concentration of polynucleotides innucleotides (M), k₁=the association rate constant (M⁻¹s⁻¹), and k₂=thedissociation rate constant (s⁻¹). Both k₁ and k₂ are unknowns in thisequation and are estimated by the practitioner. To begin the analysis,k₁ is assigned a rate constant value derived from a conventional C_(o)tanalysis and k₂ is assigned a rate constant value of zero, whichpresupposes that there was no dissociation during the reaction. (Whenk₂=0, this equation reduces to the C_(o)t equation.) Plugging the actualt values and estimated k₁ and k₂ values into the new equation, curveplotting software, such as KaleidaGraph 3.0 (Synergy Software; Reading,Pa.), can be used to generate a curve which relates percenthybridization and C_(o) across a range of concentrations tested.Examples of such curves are depicted in FIGS. 3 and 4, which superimposethe curves generated using the curve plotting software over the plotteddata points () derived from the C_(o)t analysis and depicted in FIGS. 1and 2. (For FIGS. 3 and 4, a k₁ rate constant of 16,000 M⁻¹s⁻¹ and a k₂rate constant of 0 were fitted into the equation.) As can be seen fromFIGS. 3 and 4, the curves and plotted data points in each of thesegraphs are not entirely coincident.

[0116] Finally, k, and k₂ are adjusted until the plotted data pointsfrom the experimental data are coincident with the curves plotted fromthe new equation using the curve plotting software. FIGS. 5 and 6 showthe graphs of FIGS. 3 and 4 after k₁ and k₂ have been adjusted,resulting in a curve and plotted data points () which coincide in eachgraph. (To obtain a coincident curve and plotted data points for each ofthese graphs, k₁ was adjusted to 14,500 M⁻¹s⁻¹ and k₂ was adjusted to8.33×10⁻⁴s⁻¹.) When two incubation periods are used, as in the examplerepresented in FIGS. 1-6, k₁ and k₂ must be the same for both reactionsto ensure that the amount of dissociation is being accurately accountedfor in arriving at a final association rate constant (k₁). As statedabove, one reason for testing two association periods is that theassociation and dissociation of polynucleotides tend to increase overtime of reaction. Thus, data for two distinct reaction times results ina more accurate determination of the rate of association.

[0117] Either the conventional C_(o)t analysis or the new equation andcurve plotting procedure described above may be used to determine theextent to which any particular polycationic polymer or group ofpolycationic polymers enhance the rate of association for a set ofpolynucleotides. While the latter is preferred for quantitative reasons,both methods will provide a qualitative measure of whether apolycationic polymer or group of polycationic polymers positively affectthe kinetics of an association reaction. This is demonstrated in FIG. 7,which directly compares percent of hybridization versus log of C_(o)tfor poly-L-histidine hydrochloride (♦) and no polymer (Δ) underidentical hybridization conditions based on data from Example 1 infra.It will be understood, however, that both equations are estimates ofactual association rates, which become more difficult to calculate withaccuracy as association rates increase.

F. Detection Systems

[0118] Before a target nucleic acid can be detected in a test sample, itmust be made available in the reaction mixture for hybridization to apolynucleotide probe. Many cellular disruption methods for releasingnucleic acid into a reaction mixture are well known in the art, andinclude both chemical and enzymatic methods, as well as mechanical meanssuch as ultrasonication, agitation with glass beads, grinding withabrasives and the French pressure cell. Other methods involve weakeningthe cell wall by one or more rounds of freezing and thawing or bytreatment with a lysing enzyme such as lysozyme, followed by dissolutionof the cell membrane by treatment with a strong detergent or achaotropic reagent (i.e., a reagent that disrupts hydrophobicinteractions). The lysate of these methods include organelles, proteins(including enzymes such as proteases and nucleases), carbohydrates, andlipids as well as nucleic acids, which may require further purificationof the nucleic acids.

[0119] An extraction method requiring only a single reagent to releasenucleic acids from a wide range of cellular types in a form suitable fornucleic acid hybridization without the need for subsequent purificationsteps is disclosed by Clark et al., “Method for Extracting Nucleic Acidsfrom a Wide Range of Organisms,” U.S. Pat. No. 5,786,208. Thisextraction method combines a test sample with a reagent which includes anon-ionic detergent, an optional anionic detergent and a metal chelatingagent and heats the resulting mixture at a temperature between 80° and100° C. until nucleic acids are released from the cells. Because anionicdetergents such as lithium lauryl sulfate are believed to disrupt ordenature nucleases (e.g., ribonucleases) present in the test sample,inclusion of an anionic detergent is particularly desirable when thetarget nucleic acid is RNA. (In this and other extraction methodsemploying an anionic detergent, the target nucleic acid is preferablyseparated from the anionic detergent prior to contacting the targetnucleic acid with the polycationic polymers of the present invention.)Nucleic acids are released in this method without observable destructionto cell walls, so that the liberated nucleic acids are suitable forhybridization, amplification or other genetic manipulations withoutfurther purification.

[0120] Following sample preparation, the polycationic polymers of thepresent invention may be used in a variety of detection systems,including both heterogenous and homogenous systems used to determine thepresence or amount of target nucleic acids in a sample. In aheterogenous assay, a step is required to isolate or separateprobe:target hybrids from excess probe sequences before single-strandedprobes and probe:target hybrids can be distinguished from each other.Examples of heterogenous assay systems are disclosed by, for example,Ranki et al. in U.S. Pat. No. 4,486,539 and Stabinsky in U.S. Pat. No.4,751,177. Homogenous assays on the other hand require no separationstep, thereby permitting the in solution detection of probe:targethybrids in the presence of excess probe sequences. Examples of detectionsystems which can be used in either the heterogenous or homogenoussystems are the Hybridization Protection Assay and the Adduct ProtectionAssay disclosed by Arnold et al. in U.S. Pat. No. 5,283,174, and Beckeret al., “Adduct Protection Assay,” U.S. Pat. No. 5,731,148,respectively. Other well known detection systems employ self-hybridizingprobes which incorporate interacting labels that emit differentiallydetectable signals, depending upon whether the probes are bound totarget nucleic acid or remain self-hybridized in the reaction mixture.See, e.g., Bagwell in U.S. Pat. No. 5,607,834; Tyagi et al. in U.S. Pat.No. 5,925,517; and Becker et al. in U.S. Pat. No. 6,361,945.

[0121] The Hybridization Protection Assay is a particularly preferredhomogenous detection system based on differential hydrolysis. See Arnoldet al. in U.S. Pat. No. 5,283,174; see also Arnold et al. ClinicalChemistry (1989) 35:1588-1594. In this detection system, an excess ofprobe labeled with a chemiluminescent acridinium ester is provided to atest sample for a period of time and under conditions permitting theprobe to stably hybridize to a target nucleic acid suspected of beingpresent in the test sample. Following hybridization, acridinium esterlabel associated with unhybridized probe is selectively degraded byproviding an alkaline reagent to the test sample which hydrolyzes of thephenyl ester of the acridinium ester. Label associated with hybridizedprobe is protected from hydrolysis by intercalation of the label in theduplexed molecule. Thus, the amount of acridinium ester remaining in thetest sample is proportional to the amount of hybrid and can be measuredby the chemiluminescence produced from acridinium ester labelsassociated with hybridized probe upon the addition of hydrogen peroxidefollowed by alkali. Chemiluminescence can be measured in a luminometer,including the LEADER® 450i luminometer. Useful HPA conditions andreagents are exemplified in Example 1 below.

[0122] The adduct protection assay, which is preferred when measuringhybridization kinetics, can facilitate the detection of a targetpolynucleotide by exploiting adduct formation to preferentially altersignal production from a label present on a polynucleotide probe notbound to the target polynucleotide. The assay involves the formation ofa protective micro-environment when a labeled polynucleotide probe formsa duplex with the target polynucleotide. Label associated with probebound to target polynucleotide is preferentially protected from formingan adduct with a signal altering ligand, such as sodium sulfite. Labelassociated with free probe, however, can be selectively altered in thepresence of the signal altering ligand, thereby affecting its ability toproduce a detectable signal. Examples of signal altering ligands usefulin an adduct protection assay include tetrahydrothiopene, propanethiol,benzylmercaptan, sulfite, glycol sulfite, hydrosulfite, metabisulfite,thiosulfate, thiophosphate, metaarsenite, tellurite, arsenite andthiocyanate. By introducing a signal triggering reagent which causeslabel to produce a detectable signal, the presence or amount of aprobe:target duplex in a sample can be determined. The preferred labelis a chemiluminescent reagent, such as an acridinium ester, and thepreferred signal triggering reagent is sodium hydroxide or hydrogenperoxide. Acridinium ester labels and means for their detection aredisclosed by Arnold et al. in U.S. Pat. No. 5,185,439.

[0123] Before signal triggering reagent is introduced into the sample,polycationic polymers and polynucleotides are first dissociated fromeach other in the preferred adduct protection assay. (A dissociationstep is not required for all embodiments of the present invention.)Dissociation of polycationic polymers from polynucleotides can beeffected with anionic detergents and polyanions which weaken the bindingof polycations to nucleic acids (e.g., polyglutamic acid, polyasparticacid, sodium dodecyl sulfate and polynucleotides). For this reason,where a dissociation step is to be included, the sample should excludeappreciable amounts of anionic detergents and polyanions capable ofdissociating polycationic polymers from polynucleotides prior to thedissociation step so that the enhanced association of complementarypolynucleotides in the presence of polycationic polymers can proceedunimpeded. More particularly, the total anionic charge in the reactionmixture should be less than the total cationic charge. A preferreddissociating reagent is lithium lauryl sulfate (LLS) at a finalconcentration of about 1% (w/v) in the reaction mixture.

[0124] Prior to detection, it may be desirable to increase the quantityof target nucleic acid present, and thus the sensitivity of the assay,by exposing the reaction mixture to nucleic acid amplificationconditions. Under amplification conditions, polynucleotide chainscontaining the target sequence or its complement are synthesized in atemplate-dependent manner from ribonucleoside or deoxynucleosidetriphosphates using nucleotidyltransferases known as polymerases. Thereare many amplification procedures in common use today, including thepolymerase chain reaction (PCR), Q-beta replicase, self-sustainedsequence replication (3SR), transcription-mediated amplification (TMA),nucleic acid sequence-based amplification (NASBA), ligase chain reaction(LCR), strand displacement amplification (SDA) and loop-mediatedisothermal amplification (LAMP), each of which is well known in the art.See, e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S.Pat. No. 4,683,202; Erlich et al., “Kits for Amplifying and DetectingNucleic Acid Sequences,” U.S. Pat. No. 6,197,563; Walker et al. NucleicAcids Res. (1992) 20:1691-1696; Fahy et al. PCR Methods and Applications(1991) 1:25-33; Kacian et al., U.S. Pat. No. 5,399,491; Kacian et al.,“Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,480,784;Davey et al., “Nucleic Acid Amplification Process,” U.S. Pat. No.5,554,517; Birkenmeyer et al., “Amplification of Target Nucleic AcidsUsing Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930;Marshall et al., “Amplification of RNA Sequences Using the Ligase ChainReaction,” U.S. Pat. No. 5,686,272; Walker, “Strand DisplacementAmplification,” U.S. Pat. No. 5,712,124; Notomi et al., “Process forSynthesizing Nucleic Acid,” European Patent Application No. 1 020 534A1; Dattagupta et al., “Isothermal Strand Displacement Amplification,”U.S. Pat. No. 6,214,587; and HELEN H. LEE ET AL., NUCLEIC ACIDAMPLIFICATION TECHNOLOGIES: APPLICATION TO DISEASE DIAGNOSIS (1997).

[0125] A preferred method for amplifying a target sequence istranscription-mediated amplification (TMA). See, e.g., Kacian et al. inU.S. Pat. Nos. 5,399,491 and 5,480,784 and LEE ET AL., supra, ch. 8. TMAis an isothermal amplification procedure which allows for a greater thanone billion-fold increase in copy number of the target sequence usingreverse transcriptase and RNA polymerase. The target sequence in a TMAamplification may be any type of nucleic acid, including rRNA, mRNA orDNA. TMA reaction involves converting a single-stranded target sequenceto a double-stranded DNA intermediate by reverse transcriptase in thepresence of a sense primer and an antisense primer having a 5′ RNApolymerase-specific promoter sequence (i.e., promoter-primer). Reversetranscriptase creates a DNA copy of the target sequence by extensionfrom the 3′ end of the promoter-primer in the presence of nucleosidetriphosphate substrates. Where the target sequence is RNA, the RNA inthe resulting DNA:RNA duplex is degraded by RNase H activities ofreverse transcriptase. The sense primer then binds to the DNA copy, anda new strand of DNA is synthesized from the 3′ end of the sense primerby reverse transcriptase, thereby creating a double-stranded DNAintermediate molecule. Included in this DNA intermediate is adouble-stranded promoter sequence which is recognized by RNA polymeraseand transcribed into hundreds of copies of RNA. Each of thesetranscribed RNA molecules, in turn, can be converted to adouble-stranded DNA intermediate which is used for producing additionalRNA. Thus, TMA reactions proceed exponentially. Particular parameters ofa TMA reaction, including concentrations of enzymes, primers andnucleoside triphosphates, as well as reaction times and temperatures,can be determined and adapted from what is well known in the art aboutTMA reactions without having to engage in undue experimentation.

[0126] If the detection step is preceded by an amplification step, thetarget nucleic acid is preferably isolated and purified beforeamplifying the target sequence. A wide variety of procedures forisolating and purifying a target nucleic acid are well known in the art.

[0127] A particularly preferred method for isolating and purifying atarget nucleic acid prior to amplification is disclosed by Weisberg etal. in U.S. Pat. No. 6,280,952. In this system, the capture probehybridizes to the target nucleic acid and an immobilized probehybridizes to the capture probe:target complex under differenthybridization conditions. Under a first set of hybridization conditions,hybridization of the capture probe to the target nucleic acid is favoredover hybridization of the capture probe to the immobilized probe. Thus,under this first set of conditions, the capture probe is in solutionrather than bound to a solid support, thereby maximizing theconcentration of the free capture probe and utilizing favorable liquidphase kinetics for hybridization to the target nucleic acid.Polycationic polymers of the present invention may be provided to thereaction mixture under this first set of conditions to promote rapidhybridization of the capture probe to the target nucleic acid. After thecapture probe has had sufficient time to hybridize to the target nucleicacid, a second set of hybridization conditions is imposed permitting inthe capture probe:target complex to hybridize to the immobilized probe,thereby isolating the target nucleic acid in the sample solution. Theimmobilized target nucleic acid may then be purified, and a targetsequence present in the target nucleic acid may be amplified anddetected. A purification procedure which includes one or more wash stepsis generally desirable when working with crude samples (e.g., clinical,environmental, industrial, food, water, etc.) to prevent enzymeinhibition and/or nucleic acid degradation due to substances present inthe sample.

[0128] Instrument systems for performing detection assays are well knownin the art and may be used to perform manual, semi-automated or fullyautomated assays. Some of these instrument systems are limited to directdetection (no prior amplification step), while others have thecapability of performing both amplification and detection. Theseinstrument systems may detect the formation of polynucleotide hybridsusing any of a variety of techniques known in the art including, but notlimited to, those based on light emission, mass changes, changes inconductivity or turbidity. Examples of instrument systems which could bereadily adapted to perform assays incorporating the polycationicpolymers of the present invention in order to enhance reaction ratesinclude those sold under the trade names of DTS 400 (detection only) andDTS 1600 (amplification and detection) by Gen-Probe Incorporated of SanDiego, Calif., which represent embodiments of instrument systemsdisclosed by Acosta et al., “Assay Work Station,” U.S. Pat. No.6,254,826, and by Ammann et al., “Automated Process for Isolating andAmplifying a Target Nucleic Acid Sequence,” U.S. application Ser. No.09/303,030, and International Publication No. WO 99/57561, “AutomatedDiagnostic Analyzer and Method,” each of which enjoys common ownershipherewith.

G. Kits

[0129] The present invention also contemplates detection systems in kitform. Kits of the present invention include, in an amount sufficient forat least one assay, a polynucleotide probe which preferentiallyhybridizes to a target nucleic acid sequence in a test sample underhybridization assay conditions and a synthetic polycationic polymer inan amount sufficient to increase the association rate of the probe andthe target sequence in the test sample under the hybridization assayconditions. The probe and the polymer may be combined in the same orseparate containers. Kits containing multiple probes are alsocontemplated by the present invention where the multiple probes aredesigned to target different nucleic acid sequences and may includedistinct labels which permit the probes to be differentially detected ina test sample. Kits according to the present invention may furthercomprise at least one of the following: (i) a reagent in an amountsufficient to dissociate the polymer from the probe in the test sample;(ii) one or more amplification primers for amplifying a target sequencecontained in or derived from the target nucleic acid; (iii) a captureprobe for isolating and purifying target nucleic acid present in a testsample; and (iv) if a capture probe is included, a solid supportmaterial (e.g., magnetically responsive particles) for immobilizing thecapture probe, either directly or indirectly, in a test sample. Kits ofthe present invention may further include one or more helper probes.

[0130] Typically, the kits will also include instructions recorded in atangible form (e.g., contained on paper or an electronic medium) forusing the packaged probe and polymer in a detection assay fordetermining the presence or amount of a target nucleic acid sequence ina test sample. The assay described in the written instructions mayinclude steps for isolating and purifying the target nucleic acid priorto detection with the polynucleotide probe, amplifying a target sequencecontained in the target nucleic acid, and/or dissociating the probe fromthe polymer using a dissociating reagent. The detection assay may bediagnostic for the presence of a particular virus or organism or groupof viruses or organisms, disease or condition, or it may be useful fordetermining a disease state or level of gene expression or for detectingthe presence of a mutation or polymorphism.

[0131] The various components of the detection systems may be providedin a variety of forms. For example, the probe and/or polycationicpolymer may be provided as lyophilized reagents. The lyophilizedreagents may be pre-mixed before lyophilization so that whenreconstituted they form a complete mixture with the proper ratio of eachof the components ready for use in the assay. In addition, the detectionsystems of the present invention may contain a reconstitution reagentfor reconstituting the lyophilized reagents of the kit. Preferred kitscontain lyophilized probe reagents.

[0132] Typical packaging materials include solid matrices, such asglass, plastic, paper, foil, micro-particles and the like, which arecapable of holding within fixed limits the probe, polycationic polymerand other optional reagents of the present invention. Thus, for example,the packaging materials can include glass vials used to containsub-milligram quantities of a contemplated probe or polycationicpolymer, or they can be microtiter plate wells to which probes of thepresent invention have been operatively affixed, i.e., linked so as tobe capable of participating in a procedure for detecting a targetnucleic acid sequence.

[0133] The instructions will typically indicate the reagents and/orconcentrations of reagents and at least one assay method parameter whichmight be, for example, the relative amounts of reagents to use peramount of sample. In addition, such specifics as maintenance, timeperiods, temperature and buffer conditions may also be included.

H. EXAMPLES

[0134] Examples are provided below illustrating different aspects andembodiments of the invention. Skilled artisans will appreciate thatthese examples are not intended to limit the invention to the specificembodiments described therein.

Example 1 Effect of Polycationic Polymers on Hybridization KineticsBetween Perfectly Complementary Probe and Target Sequences

[0135] This example shows the effect that various polycationic polymershad on the rate at which a polynucleotide probe and a perfectlycomplementary synthetic target sequence associated under differentcombinations of salt and temperature conditions. For this example, theprobe had the nucleotide base sequence of SEQ ID NO:1gctcgttgcgggactt(*)aacccaacat, which was synthesized to include anon-nucleotide linker (the asterik indicates the location of thenon-nucleotide linker), as disclosed by Arnold et al. in U.S. Pat. No.6,031,091. The probe was labeled with a chemiluminescent acridiniumester (standard AE), as disclosed by Arnold et al. in U.S. Pat. No.5,185,439, and measured in relative light units (RLU).

[0136] The following five polymers were tested in this example, and eachpolymer was indicated to have the noted properties by the supplier:

[0137] (i) Poly-L-lysine hydrobromide. This polymer was purchased fromFluka AG (Cat. No. 81333; Lot No. 307943/1 497) and was indicated tohave a M_(w) of between 20,000 and 30,000 Da (“Low M_(w)Poly-L-lysine”);

[0138] (ii) Poly-L-lysine hydrobromide. This polymer was purchased fromFluka AG (Cat. No. 81355; Lot No. 299299/1 1093) and was indicated tohave a M_(w) of between 150,000 and 300,000 Da (“High M_(w)Poly-L-lysine”);

[0139] (iii) Poly (lys, tyr) 4:1. This polymer was purchased from SigmaChemical Company (Product No. P 4659; Lot No. 81H5520) and was indicatedto have a M_(w) of 24,600 Da (visible) and a degree of polymerization of123 (visible);

[0140] (iv) Poly-L-histidine hydrochloride. This polymer was purchasedfrom Sigma Chemical Company (Product No. P 2534; Lot No. 118H5905) andwas indicated to have a M_(w) of 15,800 Da (using low angle laser lightscattering) and a degree of polymerization of 91 (using low angle laserlight scattering);

[0141] (v) Poly-L-arginine hydrochloride. This polymer was purchasedfrom Sigma Chemical Company (Product No. P 4663; Lot No. 87H5903) andwas indicated to have a M_(w) of 11,800 Da (visible) and 8,400 Da (usinglow angle laser light scattering), a degree of polymerization of 43(using low angle laser light scattering), and a M_(w)/M_(n) of 1.25(using low angle laser light scattering with size exclusionchromatography); and

[0142] (vi) Hexadimethrine bromide. This polymer(1,5-dimethyl-1,5-diazaundecamethylene polymethobrornide) was purchasedfrom Sigma Chemical Company (Product No. H 9268; Lot No. 50K3672).

[0143] For each polymer tested, a total of 40 12×75 mm polypropylenetubes (Gen-Probe Incorporated; Cat. No. 2440) were set up, and each tubereceived 0.5 fmol probe and 20 μM polymer dissolved in 40 μlhybridization buffer. A no polymer control set was also tested in 4012×75 mm polypropylene tubes (Gen-Probe Incorporated; Cat. No. 2440)which received only 0.5 fmol probe dissolved in 40 μl hybridizationbuffer. The hybridization buffer was either a high salt hybridizationbuffer made up of 200 mM lithium succinate, pH 5.1, 0.70 M LiCl and 0.2%(v/v) TRITON® X-100 or a low salt hybridization buffer made up of 200 mMlithium succinate, pH 5.1, 100 mM LiCl and 0.2% (v/v) TRITON® X-100. Thetubes were divided into two groups of 20 tubes each and all tubes werepre-heated to 40° C. or 60° C. (consistent with the hybridizationtemperature indicated below) for two minutes in a circulating water bath(Lauda Dr. R. Wobser GmbH & Co. KG, Lauda-Koenigshofen, Germany; ModelNo. E100). With the exception of 4 control tubes for determiningbackground RLU values in each group, 40 μl filtered water (MilliporeCorporation; Bedford, Mass.; Milli-Q UF Plus; Cat. No. ZD5311595)containing synthetic RNA target sequence was added to the tubes of bothgroups and the tubes were mixed briefly by hand. The amount of targetsequence in the 16 target-containing tubes of each group ranged (inincreasing concentrations) from as low as 0.05 fmol to as high as100,000 fmol. Precise target sequence concentrations for individualtubes are indicated in Tables 1 and 2 below. The target sequence used inthis example was the RNA complement of the probe (SEQ ID NO:2auguuggguuaagucccgcaacgagc).

[0144] After adding the target sequence, the tubes of groups one and twowere heated to 40° C. or 60° C. in a circulating water bath (Lauda Dr.R. Wobser GmbH & Co. KG; Model No. E100) to allow hybridization of theprobe to the target sequence. The incubation time was three minutes forthe tubes of group one and eight minutes for the tubes of group two.Following hybridization, lithium lauryl sulfate (LLS) was added to allof the tubes to dissociate polymer from probe. The final concentrationof the LLS was 1% (w/v) for all tubes. The tubes were then placed in anice water bath for approximately five minutes to arrest thehybridization reaction. All tubes were then analyzed in a LEADER® 50luminometer (Gen-Probe Incorporated; Cat. No. 3100) equipped withautomatic injection of detection reagents comprised of Detect Reagent I,which contained 0.14 M sodium sulfite and 0.042 M sodium borate, andDetect Reagent II, which contained 1.5 M sodium hydroxide and 0.12%(v/v) sodium peroxide. A 12 second pause was introduced betweeninjections of the two Detect Reagents.

[0145] To determine the rate of association for no polymer and eachpolymer tested, the novel equation and method for calculating rateconstants described in the Association Kinetics section supra werefollowed. Following this approach, the first step was to determine theC_(o)t value and the percent hybridization for each concentration oftarget tested for each incubation period. Percent hybridizations weredetermined by dividing the net RLU for each target concentration by theRLU observed at high target concentrations where hybridization wascomplete. The net RLU for each target concentration was determined bysubtracting the average background RLU observed in the four blank tubesfor each test performed from the raw RLU for each target concentration.Each of these values is set forth in Tables 1 and 2 below for the nopolymer 3 and 8 minute incubations tested in the low salt hybridizationbuffer at 40° C. (The values determined for each of the polymers tested,as well as no polymer tested in the high salt hybridization buffer at60° C., are not shown in Tables 1 and 2.) The percent hybridizationversus the log of C_(o)t data points ( ) were then plotted on thegraphs, which are shown in FIGS. 1 and 2 for the no polymer 3 and 8minute incubations.

[0146] Predicted graphs of percent hybridization versus the log ofC_(o)t were then determined based on percent hybridization valuescalculated using the following equation: %hybridization=100(1−K)(1−exp−(k₁C_(o)+k₂)t), where K=k₂/(C_(o)k₁+k₂),t=time (s), C_(o)=initial concentration of polynucleotides innucleotides (M), k₁=the association rate constant (M⁻¹s⁻¹), and k₂=thedissociation rate constant (s⁻¹). Being unknowns, k₁ and k₂ wereinitially assigned estimated values. The estimated k, values weredetermined using the conventional C_(o)t analysis discussed in theAssociation Kinetics section supra (e.g., k, was determined to be 16,000M⁻¹s⁻¹ by conventional C_(o)t analysis for the no polymer testsillustrated) and the estimated value of k₂ was always zero. Afterplugging the actual t values and the estimated k₁ and k₂ values for eachtest performed into the equation above, KaleidaGraph 3.0 software wasused to generate curves which related percent hybridization and C_(o)across the range of target concentrations tested. The curves generatedin this manner for the no polymer 3 and 8 minute incubations aredepicted in FIGS. 3 and 4, where the predicted curves are shownsuperimposed over the graphs plotted from the experimental data andshown in FIGS. 1 and 2. The greater discrepancy between the predictedcurve and the plotted data points observed at 8 minutes versus 3 minutesfor each test indicated a k₂ that was greater than zero.

[0147] For this reason, the k₁ and k₂ values were adjusted for each testuntil the data points plotted from the experimental data were coincidentwith the curves plotted from the above new equation using the curveplotting software, as shown in FIGS. 5 and 6. Thus, the final k₁ and k₂values for each test were the same for both incubation times, therebyensuring that the concomitant dissociation of polynucleotides over timewas being accounted for in the k, determination. Rate constantsdetermined in this manner are set forth in Table 3 below for no polymerand each of the polymers tested. TABLE 1 C_(o)t Values and PercentHybridization for Various Concentrations of Target After a Three MinuteIncubation in a Low Salt Hybridization Buffer at 40° C. in the Absenceof Polymer Target C_(o)t Percent (fmol) (M⁻¹s⁻¹) Raw RLU Net RLUHybridization 0 0 1526 0 0 1 5.85 × 10⁻⁸ 1607 81 0.15 2 1.17 × 10⁻⁷ 27271201 2.26 5 2.93 × 10⁻⁷ 1791 265 0.50 10 5.85 × 10⁻⁷ 2026 500 0.94 201.17 × 10⁻⁶ 2289 763 1.44 50 2.93 × 10⁻⁶ 3526 2000 3.77 100 5.85 × 10⁻⁶5730 4204 7.93 200 1.17 × 10⁻⁵ 9850 8324 15.69 500 2.93 × 10⁻⁵ 19,54518,019 33.97 1000 5.85 × 10⁻⁵ 31,340 29,814 56.21 2000 1.17 × 10⁻⁴42,403 40,877 77.07 5000 2.93 × 10⁻⁴ 51,067 49,541 93.40 20,000 1.17 ×10⁻³ 53,460 51,934 97.92 50,000 2.93 × 10⁻³ 55,629 54,103 102.01 100,0005.85 × 10⁻³ 54,607 53,081 100.08

[0148] TABLE 2 C_(o)t Values and Percent Hybridization for VariousConcentrations of Target After An Eight Minute Incubation in a Low SaltHybridization Buffer at 40° C. in the Absence of Polymer Target C_(o)tPercent (fmol) (M⁻¹s⁻¹) Raw RLU Net RLU Hybridization 0 0 1641 0 0 11.56 × 10⁻⁷ 1605 −36 −0.07 2 3.12 × 10⁻⁷ 1705 64 0.13 5  7.8 × 10⁻⁷ 1871230 0.47 10 1.56 × 10⁻⁶ 2484 843 1.73 20 3.12 × 10⁻⁶ 2963 1322 2.71 50 7.8 × 10⁻⁶ 5123 3482 7.13 100 1.56 × 10⁻⁵ 8240 6599 13.51 200 3.12 ×10⁻⁵ 13,901 12,260 25.11 500  7.8 × 10⁻⁵ 27,400 25,759 52.75 1000 1.56 ×10⁻⁴ 37,532 35,891 73.50 2000 3.12 × 10⁻⁴ 46,501 44,860 91.87 5000  7.8× 10⁻⁴ 50,159 48,518 99.36 10,000 1.56 × 10⁻³ 49,613 47,972 98.24 50,000 7.8 × 10⁻³ 50,739 40,098 100.54 100,000 1.56 × 10⁻² 50,434 48,793 99.92

[0149] TABLE 3 Rate Constant of Probe in the Presence of VariousPolymers and No Polymer Under Different Temperature and SaltConcentration Conditions Salt Rate Temperature Concentration ConstantPolymer (° C.) (M) (M⁻¹s⁻¹) No Polymer 60 0.45  5.7 × 10⁴ No Polymer 400.15 1.45 × 10⁴ Poly-_(L)-lysine hydrobromide 60 0.45   1 × 10⁵ (LowM_(w) Poly-_(L)-lysine) Poly-_(L)-lysine hydrobromide 60 0.45  5.7 × 10⁴(High M_(w) Poly-_(L)-lysine) Poly-_(L)-lysine hydrobromide 40 0.15   ≧2× 10⁷ (High M_(w) Poly-_(L)-lysine) Poly (lys, tyr) 4:1 60 0.45   6 ×10⁵ Poly-_(L)-histidine hydro- 60 0.45   5 × 10⁶ chloridePoly-_(L)-arginine hydro- 60 0.45   5 × 10⁶ chloride Poly-_(L)-argininehydro- 40 0.15   ≧2 × 10⁷ chloride Hexadimethrine bromide 60 0.15   3 ×10⁶

[0150] The results of this experiment demonstrate that the presence ofpolycationic polymers in a reaction mixture can significantly enhancethe rate of hybridization between a polynucleotide probe and a perfectlycomplementary target sequence. The results of this experiment furthershow that this enhanced rate of hybridization can be achieved usingpolycationic polymers under conditions promoting hybridization (e.g.,high salt conditions).

Example 2 Effect of Polycationic Polymers on Hybridization KineticsBetween Probe and Mutant Target Sequences

[0151] This example shows the effect that various polycationic polymershad on the rate at which a polynucleotide probe and a mutant targetsequence associated under different combinations of salt and temperatureconditions. The reagents, concentrations, times, conditions, tubes andinstruments used in this example were identical to those of Example 1above, except that a single base mismatch between the probe and thesynthetic RNA target sequence was introduced at the fourth nucleotideposition reading from the 5′ end of the probe sequence. This wasachieved by using the same target sequence as Example 1 and altering theprobe to have the nucleotide base sequence of SEQ ID NO:3gctgttgcgggactt(*)aacccaacat (the asterik indicates the location of anon-nucleotide linker). The rates listed below were determined in thesame manner detailed above in Example 1. TABLE 4 Rate Constant of ProbeContaining a Single-Base Mismatch in the Presence of Various Polymersand No Polymer Under Identical Temperature and Salt ConcentrationConditions Salt Temperature Concentration Rate Polymer (° C.) (M)(M⁻¹s⁻¹) No Polymer 60 0.45   6 × 10³ Poly-_(L)-lysine hydrobromide 600.45 1.5 × 10⁴ (Low M_(w) Poly-_(L)-lysine) Poly (lys, tyr) 4:1 60 0.45  1 × 10⁵ Poly-_(L)-histidine hydro- 60 0.45 1.5 × 10⁶ chloridePoly-_(L)-arginine hydro- 60 0.45   6 × 10⁶ chloride Poly-_(L)-argininehydro- 40 0.15   6 × 10⁶ chloride Hexadimethrine bromide 60 0.15   6 ×10⁶

[0152] This experiment demonstrated that the presence of somepolycationic polymers in a reaction mixture can enhance the rate ofhybridization between a polynucleotide probe and a mutant targetsequence having a single base mismatch to a sufficient degree to allowfor the detection of different subtypes in a reaction mixture. Here, theresults indicate that the poly-L-arginine hydrochloride (high saltconcentration) and hexadimethrine bromide (low salt concentration)polymers tolerated the mismatch, whereas the remainder of the polymerstested were sensitive to the mismatch. The sensitivity of theseremaining polymers suggests that they would enhance the rate ofassociation between a probe and its complementary sequence while at thesame time allowing for single base mismatch discrimination.

[0153] While the present invention has been described and shown inconsiderable detail with reference to certain preferred embodiments,those skilled in the art will readily appreciate other embodiments ofthe present invention. Accordingly, the present invention is deemed toinclude all modifications and variations encompassed within the spiritand scope of the following appended claims.

1 3 1 26 DNA Artificial Sequence Synthetic Construct 1 gctcgttgcgggacttaacc caacat 26 2 26 RNA Artificial Sequence Synthetic Construct 2auguuggguu aagucccgca acgagc 26 3 25 DNA Artificial Sequence SyntheticConstruct 3 gctgttgcgg gacttaaccc aacat 25

What I claim is:
 1. A kit comprising: a polynucleotide probe whichpreferentially hybridizes to a target nucleic acid present in a testsample under a first set of hybridization conditions; a syntheticpolycationic polymer in an amount sufficient to increase the associationrate of said probe and said target nucleic acid in said sample undersaid first set of hybridization conditions; and a dissociating reagentfor dissociating said polymer from said probe and said target nucleicacid in said sample.
 2. The kit of claim 1, wherein the cationicmonomers comprising said polymer are in molar excess of the phosphategroups of said probe.
 3. The kit of claim 1, wherein said polymer iscopolymer.
 4. The kit of claim 1, wherein said polymer is a graftcopolymer.
 5. The kit of claim 1, wherein said polymer has a delocalizedcharge.
 6. The kit of claim 1, wherein said polymer has a weight averagemolecular weight of less than about 300,000 Da.
 7. The kit of claim 1,wherein said probe includes multiple interacting labels and comprisesfirst and second base regions which hybridize to each other under saidfirst set of hybridization conditions in the absence of said targetsequence, wherein said labels interact with each other to produce afirst detectable signal when said probe is not hybridized to said targetsequence and a second detectable signal when said probe is hybridized tosaid target sequence, and wherein said first and second signals aredetectably different from each other.
 8. The kit of claim 7, whereinsaid probe includes a third base region which hybridizes to said targetsequence under said first set of hybridization conditions, and whereinsaid third base region is distinct from said first and second baseregions or said third base region partially or fully overlaps at leastone of said first and second base regions of said probe.
 9. The kit ofclaim 1, wherein said probe is a polyanion.
 10. The kit of claim 9,wherein said probe further includes at least one of a cationic group anda nonionic group.
 11. The kit of claim 9, wherein the distance betweenadjacent cationic monomers of said polymer approximates the distancebetween adjacent phosphate groups of said probe.
 12. The kit of claims1, where said target sequence comprises RNA.
 13. The kit of claim 12,wherein said RNA is ribosomal RNA.
 14. The kit of claim 12, wherein saidRNA is messenger RNA.
 15. The kit of claim 1, wherein said dissociatingreagent is at least one of a polyanion or an anionic detergent.
 16. Thekit of claim 1 further comprising written instructions for performing anassay to determine the presence or absence of said target sequence insaid sample as an indication of the presence or absence of a virus ororganism or members of a group of viruses or organisms in said sample.17. The kit of claim 16, wherein said written instructions specifyhybridization conditions which include a temperature of at least about40° C. and a salt concentration of at least about 5 mM monovalentcations or an equivalent salt concentration containing multivalentcations.
 18. The kit of claim 17, wherein the temperature specified bysaid written instruction is up to about 60° C.
 19. The kit of claim 16,wherein said written instructions specifiy hybridization conditionswhich include a temperature of at least about 40° C. and a saltconcentration of at least about 150 mM monovalent cations or anequivalent salt concentration containing multivalent cations.
 20. Thekit of claim 19, wherein the temperature specified by said writteninstructions is up to about 60° C.
 21. The kit of claim 16, wherein saidprobe includes a label.
 22. The kit of claim 1 further comprising acapture probe having a base region which stably hybridizes to a baseregion present in said target nucleic acid under a second set ofhybridization conditions, wherein said first and second hybridizationconditions may be the same or different, and wherein said capture probestably hybridizes to said target nucleic acid under said first set ofhybridization conditions.
 23. The kit of claim 1 further comprising oneor more amplification primers.